Inpp5e is crucial for photoreceptor outer segment maintenance.

Rods and cones are functionally and morphologically distinct. We previously identified N-myc downstream-regulated gene 1b (ndrg1b) in carp as a cone-specific gene. Here, we show that NDRG1b and its paralog, NDRG1a-1, contribute to photoreceptor outer segment (OS) formation in zebrafish. In adult zebrafish photoreceptors, NDRG1a-1 was localized in the entire cone plasma membranes, and also in rod plasma membranes except its OS. NDRG1b was expressed specifically in cones in the entire plasma membranes. In a developing retina, NDRG1a-1 was expressed in the photoreceptor layer, and NDRG1b in the photoreceptor layer plus inner nuclear layer. Based on our primary knockdown study suggesting that both proteins are involved in normal rod and cone OS development, NDRG1a-1 was overexpressed or NDRG1b was ectopically expressed in rods. These forced-expression studies in the transgenic fish confirmed the effect of these proteins on the OS morphology: rod OS morphology changed from cylindrical to tapered shape. These taper-shaped rod OSs were not stained with N,N’-didansyl cystine that effectively labels infolded membrane structure of cone OS. The result shows that rod OS membrane structure is preserved in these taper-shaped OSs and therefore, suggests that tapered OS morphology is not related to the infolded membrane structure in cone OS.

PurposeJeune's Asphyxiating Thoracic Dystrophy (JATD) is an autosomal recessive disorder with symptoms of retinal degeneration, kidney cysts, and chondrodysplasia and results from mutations in the ift80 gene. This study was conducted to characterize zebrafish lacking ift80 function for photoreceptor degeneration and defects in ciliogenesis in order to establish zebrafish as a vertebrate model for visual dysfunction in JATD and to determine if ift80 interacts genetically with Bardet-Biedl Syndrome (BBS) genes.MethodsZebrafish were injected with morpholinos (MOs) targeted to the ift80 gene. Retinas were analyzed by histology, transmission electron microscopy, and immunohistochemistry. Ear and kidney cilia were analyzed by whole-mount immunostaining. Intraflagellar Transport (IFT) particle composition was analyzed by Western blotting. Genetic interactions were tested by co-injection of MOs against ift80 and bbs4 or bbs8 followed by in situ hybridization.ResultsZebrafish lacking ift80 function exhibited defects in photoreceptor outer segment formation, and photoreceptor death. Staining with opsin antibodies revealed opsin mislocalization in both rods and cones. Ultrastructural analysis showed abnormal disk stacking and shortened photoreceptor outer segments. The kinocilia of the ear and motile cilia in the kidney were shorter and reduced in number. Western blotting revealed a slight increase in the stability of other IFT proteins. Co-injection of MOs against ift80 and BBS genes led to convergent-extension defects.ConclusionsZebrafish lacking ift80 exhibited defects characteristic of Jeune's Asphyxiating Thoracic Dystrophy. Because the developing outer segments degenerated, Ift80 could possibly act as a maintenance factor for the IFT particle.

Bardet-Biedl syndrome (BBS) is a pleiotropic disorder characterized by obesity, polydactyly, and retinal degeneration, leading to blindness by the second decade of life. The blindness is caused by death of photoreceptor cells. For the purpose of phototransduction, photoreceptor cells in the retina have evolved elaborate cilia, termed the outer segments, and the structure and function of these outer segments greatly differ from that of primary cilia. The BBSome is a multi-protein complex specializing in ciliary trafficking, and it consists of BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9, and BBIP1. In this thesis, we examine the role of the BBSome in the formation and maintenance of the photoreceptor outer segment, and the efficacy of gene replacement therapies when BBSome function is restored in photoreceptors at different stages of degeneration. First, we found that the principles for BBSome recruitment to cilia differ between primary cilia and photoreceptor cilia, supporting the existence of tissue-specific mechanisms. Then, we showed that unlike primary cilia, whose formation do not require the BBSome, the proper morphogenesis of photoreceptor outer segments depends on BBSome function. Furthermore, we demonstrated that inactivation of BBSome function in adult mice causes retinal degeneration, suggesting that the BBSome is continuously required for maintenance of the outer segment. Outside of the role of the BBSome in cilia, we found that loss of BBSome function greatly reduces synaptic density between the photoreceptors and the inner neurons, supporting the growing consensus that the role of the BBSome is not limited to cilia. In addition to reduced number of synapses, BBS mutant mice also have misplaced synaptic connections within the photoreceptor layer, outside of their usual positions in the outer plexiform layer. These misplaced synaptic connections in BBS mutant mice are associated with intruding horizontal cells processes. The absence of this phenotype in mice up to three-months of age with photoreceptor-specific deletion of Bbs8, which have significant retinal degeneration, shows that the aberrant extension of horizontal cell processes in the retina is not merely a consequence to retinal degeneration. Instead, this evidence supports a cell-autonomous role of the BBSome in horizontal cells, in addition to a role in photoreceptor cells, which are typically the major target for gene therapy treatment of blindness. Lastly, we studied age-dependent rescue of retinal degeneration in a mouse line in which Bbs8 expression, and by extension BBSome function, can be activated by a removable gene trap to examine the effectiveness of gene replacement therapies. The restoration of BBSome function during earlier stages of retinal degeneration in BBS fully ameliorates retinal phenotypes, permitting the regeneration of outer segments with near normal structure. Restoration of BBSome function at later stages, however, improves the functions of rescued photoreceptors, but these photoreceptors regenerate poorly shaped outer segments and die over time. We identified the regeneration of normal outer segments to be a predictor to long-term rescue success. However, the ability for the photoreceptors to be rescued declines as retinal degeneration progresses, rendering the photoreceptors in late-stage degeneration un-rescuable. Together, this work illustrates that the BBSome functions in the outer segment morphogenesis pathway, establishes the reliance of photoreceptor survival on their elaborate outer segments, and demonstrates that effective gene therapy is confined to a limited time window during which photoreceptors can fully recover outer segment structure and function.

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in signal transduction. Cilia are anchored inside the cell through basal bodies (BBs), modified centrioles also acting as microtubule-organization centers. Photoreceptors (PRs) are sensory neurons, whose primary cilium forms a highly specialized compartment called the outer segment (OS) responsible for sensing incoming light. Thus, ciliopathies often present with retinal degeneration. Mutations in KIAA0586/TALPID3 (TA3) cause Joubert syndrome, in which 30% of affected individuals develop retinal involvement. To elucidate the function of TALPID3 in PRs, we studied talpid3 zebrafish mutants and identified a progressive retinal degeneration phenotype. The majority of PRs lack OS development due to defects in BB positioning and docking at the apical cell surface. Intracellular accumulation of the photopigment opsin leads to PR cell death of moderate severity. Electroretinograms demonstrate severe visual impairement. A small subset of PRs display normally docked BBs and extended OSs through rescue by maternally-deposited Talpid3. While localization of the small GTPase Rab8a, which plays an important role in BB docking, appears unaffected in talpid3−/− PRs, overexpression of constitutively active Rab8a rescues OS formation, indicating that the role of Ta3 in early ciliogenesis lies upstream of Rab8a activation in PRs.

To determine the specific location of the initial lesion in acute retinal pigment epitheliitis. Four patients diagnosed with acute retinal pigment epitheliitis were studied. Fundus photographs, fluorescein angiography and indocyanine green angiography, and spectral-domain optical coherence tomography findings were reviewed. Four healthy young patients presented with acute onset of unilateral decreased vision. Ophthalmoscopy showed macular pigment mottling with surrounding yellow hypopigmented areas at the level of the retinal pigment epithelium (RPE). Fluorescein angiography revealed transmission hyperfluorescence. Early-phase and midphase indocyanine green angiography images showed a patchy macular hyperfluorescence. At late phase of indocyanine green angiography, a hyperfluorescent halo with a cockadelike appearance of the macular area was observed. Spectral-domain optical coherence tomography showed a disruption of the photoreceptors' inner segment and outer segment interface associated with a wider disruption of the RPE inner band. These disrupted lines were replaced by a dome-shaped highly reflective lesion involving the RPE inner layer, the photoreceptors' inner segment and outer segment layers, and, in two cases, the outer nuclear layer. With time, indocyanine green angiography showed resolution of the observed lesions. Spectral-domain optical coherence tomography showed restored and continuous inner segment and outer segment layers and RPE inner band. Spectral-domain optical coherence tomography findings suggest that the initial lesion in acute retinal pigment epitheliitis is located at the junction between the photoreceptor outer segments and the apical side of the RPE cells. Indocyanine green angiography and spectral-domain optical coherence tomography show that the RPE appears to be more widely involved than the neurosensory retina.

During embryonic development, the rudimentary layers that will become the pigment epithelium (PE) and the neural retina are brought into close proximity upon the collapse of the optic vesicles. At this developmental stage, the retina is morphologically undifferentiated and photoreceptor outer segments have not yet begun to form. The close apposition of the PE and photoreceptors prior to the time when outer segments first appear raises the possibility that the PE could be a source of signals that induce or regulate photoreceptor development and outer segment elaboration. Also, the observation that outer segment development is limited in the absence of the PE in most species suggests that interactions between these two cell types may be of fundamental importance for the structural and functional maturation of photoreceptors [1, 2, 3, 5, 13, 16, 19, 20, 26].

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt-Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration.

Macular telangiectasia Type 2 (MacTel 2) is a bilateral disease characterized by neurodegenerative and vascular changes affecting the fovea.1 The occurrence of subretinal neovascularization in MacTel 2 is rather rare but constitutes a vision-threatening complication.2 We describe longitudinal multimodal imaging features of a novel variant of bacillary layer detachment (BLD, outer retinal split at the level of the photoreceptor inner segment myoid)3 and termed “hemorrhagic BLD” in a case of subretinal neovascularization associated with MacTel 2. This report followed the tenets of the Declaration of Helsinki. A 55-year-old woman presented with proliferative MacTel 2. Fundus photography showed active yellowish neovascular membrane with subretinal hemorrhages (Figure 1A). Optical coherence tomography (OCT) angiography segmented at the level of the outer retina confirmed subretinal neovascularization (Figure 1B). Optical coherence tomography disclosed a parafoveal outer retinal cyst consistent with BLD and adjacent subretinal fluid (Figure 1, C–E). The higher optical intensity in the BLD compared with that of subretinal fluid and its colocalization with subretinal hemorrhage on fundus photography suggested heme accumulation in the BLD. Eye-tracked follow-up OCT performed one month after a single intravitreal injection of ranibizumab (0.5 mg/0.05 mL) demonstrated significant regression of the BLD (Figure 1, F and G). Persistent focal attenuations of the ellipsoid and interdigitation zones were noted.Fig. 1.: Multimodal imaging features of hemorrhagic bacillary layer detachment. A. Color fundus photography shows typical features of macular telangiectasia Type 2 including retinal graying, crystalline deposits, ectatic capillaries, and hyperpigmentation foci. There is an active yellowish neovascular membrane (white arrowhead) with subretinal hemorrhages. B. Optical coherence tomography angiography segmented from the outer retina to the choriocapillaris shows a subretinal neovascular membrane (white arrowhead). The inset shows segmentation lines of the en face optical coherence tomography angiography image with red flow signal overlay. C–E. The (C) is a magnified view of the BLD on OCT as illustrated in (E). The intraretinal split occurs at the level of the hyporeflective myoid zone (green arrowheads). The ellipsoid zone (orange arrowhead) of the attached retina is continuous with a faint hyperreflective line (orange arrowhead) that likely represents split photoreceptor inner segments and outer segments remaining adherent to the retinal pigment epithelium/Bruch membrane complex (yellow arrowhead). The external limiting membrane (blue arrowheads) is visible over the top of the BLD (withe arrowhead). Note the dense hyperreflective material within the BLD that corresponds to heme accumulation and may interfere with accurate delineation of the outer retinal layers. The (D) is the near infrared reflectance image with the green line indicating the position of the OCT scan. Vertical OCT scan (E) shows parafoveal outer retinal cyst consistent with BLD (white arrowhead) and adjacent subretinal fluid (asterisk). F and G. Eye-tracked follow-up near infrared reflectance image (F) and OCT scan (G) performed 1 month after an intravitreal injection of ranibizumab shows dramatic resolution of the BLD. The external limiting membrane is intact. Note the persistent focal attenuations of the ellipsoid and interdigitation zones (between red arrowheads).To the best of our knowledge, hemorrhagic BLD has not been associated with proliferative MacTel 2, although similar findings coined “atypical outer retinal fluid accumulation” have been reported in neovascular age-related macular degeneration.4 Intense leakage of fluid from the subretinal neovascularization and modulation of the adherence between photoreceptor outer segments and retinal pigment epithelium by the subretinal hemorrhage may have induced BLD development.5 In fact, fibrin interdigitation between photoreceptor outer segments has been documented after experimental subretinal hemorrhages and may explain the presumed photoreceptor fracture under hydrostatic pressure effect.

The photoreceptor outer segment (OS) is a unique modification of the primary cilium, specialized for light perception. Being homologous organelles, the primary cilium and the OS share common building blocks and molecular machinery to construct and maintain them. The OS, however, has several unique structural features that are not seen in primary cilia. Although these unique features of the OS have been well documented, their implications in protein localization have been under-appreciated. In this review, we compare the structural properties of the primary cilium and the OS, and propose a hypothesis that the OS can act as a sink for membrane proteins. We further discuss the implications of this hypothesis in polarized protein localization in photoreceptors and mechanisms of photoreceptor degeneration in retinal ciliopathies.

Protein kinase C (PKC) has been implicated in regulating several proteins involved in phototransduction. This contribution characterizes the biochemical and immunological properties of PKC isozyme(s) in the photoreceptor outer segment. Activity measurements revealed that at least 85% of the PKC in this specialized compartment belongs to the subfamily of Ca2+-regulated (conventional) PKCs. Of the known Ca2+-dependent PKCs, only PKC alpha was immunodetected by western blot analysis of rod outer segment proteins. However, the ratio of immunoreactivity to enzyme activity for rod outer segment PKC was no more than 40% of that for brain PKC, using antibodies against conventional PKCs. Therefore, at least half the Ca2+/lipid-stimulated activity in rod outer segment preparations cannot be accounted for by the known isozymes, suggesting the presence of a previously uncharacterized isozyme. Despite extensive tests using a variety of antibodies against different domains of PKC alpha, PKC alpha could not be detected in rod outer segments by immunofluorescence of retinal sections. In summary, our data reveal that most of the PKC in photoreceptor outer segments is of the conventional type and that most, if not all, of this conventional PKC activity comes from a novel isozyme(s).

Dominant mutations in Arl3, linked to inherited retinal dystrophy, disrupt the active Arl3-GTP ciliary gradient and cause a defect in rod photoreceptor nuclear migration that can be rescued by elevating ciliary Arl3 activity or reducing aberrant non-ciliary Arl3 activity.

Light-related morphological changes of outer segment membranes from lamellae to tubules and two kinds of wavy configurations in the frog visual cells

Longitudinal Diffusion in Retinal Rod and Cone Outer Segment Cytoplasm: The Consequence of Cell Structure

AimsTo evaluate the recovery of retinal function and the thicknesses of the photoreceptor inner (IS) and outer segment (OS) layers after a reattachment of a rhegmatogenous retinal detachment (RRD).Methods49 eyes...

Light microscopic autoradiography and electron microscopy were used to examine outer segment renewal and the development of photoreceptors and pigment epithelium in the larval Xenopus retina. Following the injection of [3H]-leucine at stages 37/38–40 (when outer segments first develop) or 53–54 (when rod outer segments (r. o. s.) attain adult length), a band of label accumulated at the base of r. o. s. and was displaced sclerally with time, whereas label was diffusely distributed in cone outer segments (c. o. s.). By taking into account the change in shape of r. o. s. from conical to cylindrical around stage 46, and calculating outer segment growth (determined from the rate of band displacement) as volume of material added with time, we found a constant rate of membrane addition (1.59 μm/day) from the time of initial outer segment formation. The changes observed in r. o. s. length therefore indicate variations in the rate of disk shedding and phagocytosis, which is minimal before stage 46 and rises to 1.19 μm/day after stages 53–54. Ultrastructural observation showed that although all photoreceptor outer segments form by the repeated evagination of the plasma membrane of the connecting cilium, r. o. s. and c. o. s. are distinguishable by differences in membrane appearance even before they develop divergent membrane topologies. Fibrous granules near the basal body of young receptors may be precursors to the elongating ciliary microtubules. Clusters of cisternae observed near the ciliary base in photoreceptor inner segments may represent a stage in the transport of newly-synthesized opsin to the outer segment base.