Shigeru Sato

Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation

Exquisitely precise synapse formation is crucial for the mammalian CNS to function correctly. Retinal photoreceptors transfer information to bipolar and horizontal cells at a specialized synapse, the ribbon synapse. We identified pikachurin, an extracellular matrix–like retinal protein, and observed that it localized to the synaptic cleft in the photoreceptor ribbon synapse. Pikachurin null-mutant mice showed improper apposition of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, resulting in alterations in synaptic signal transmission and visual function. Pikachurin colocalized with both dystrophin and dystroglycan at the ribbon synapses. Furthermore, we observed direct biochemical interactions between pikachurin and dystroglycan. Together, our results identify pikachurin as a dystroglycan-interacting protein and demonstrate that it has an essential role in the precise interactions between the photoreceptor ribbon synapse and the bipolar dendrites. This may also advance our understanding of the molecular mechanisms underlying the retinal electrophysiological abnormalities observed in muscular dystrophy patients.

Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival

Cilia function as cell sensors in many organs, and their disorders are referred to as “ciliopathies.” Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors.

Blimp1 Suppresses Chx10 Expression in Differentiating Retinal Photoreceptor Precursors to Ensure Proper Photoreceptor Development

The zinc finger transcription factor Blimp1 plays fundamentally important roles in many cell lineages and in the early development of several cell types, including B and T lymphocytes and germ cells. Although Blimp1 expression in developing retinal photoreceptor cells has been reported, its function remains unclear. We identified Blimp1 as a downstream factor of Otx2, which plays an essential role in photoreceptor cell fate determination. To investigate Blimp1 function in the mouse retina, we ablated Blimp1 in the developing retina by conditional gene targeting. In the Blimp1 conditional knockout (CKO) retina, the number of photoreceptor cells was markedly reduced in the differentiated retina. We found that the numbers of both bipolar-like cells and proliferating retinal cells increased noticeably, with ectopic localizations in the postnatal developing retina. In contrast, a reduction of the number of photoreceptor precursors was observed during development. Forced expression of Blimp1 by in vivo electroporation suppressed bipolar cell genesis in the developing retina. Multiple genes involved in bipolar development, including Chx10, were upregulated in the Blimp1 CKO retina. Furthermore, we showed that Blimp1 can bind to the Chx10 enhancer and repress Chx10 enhancer activity. These results suggest that Blimp1 plays a crucial role in photoreceptor development by repressing genes involved in bipolar cell fate specification and retinal cell proliferation in differentiating photoreceptor precursors.

Severe Intraocular Inflammation after Intravitreal Injection of Bevacizumab

PURPOSE To report 5 cases of severe intraocular inflammation that developed after an intravitreal injection of the same lot of bevacizumab. DESIGN Retrospective case series. PARTICIPANTS Patients treated with an intravitreal injection of bevacizumab (lot B3003B01). METHODS The clinical charts of 35 eyes of 35 consecutive patients who were treated with intravitreal injection of lot B3003B01 bevacizumab from December 18, 2008, through January 20, 2009, were reviewed. MAIN OUTCOME MEASURES Incidence of intraocular inflammation, results of bacterial cultures, best-corrected visual acuity (BCVA), and endothelial cell density. RESULTS Five (14.3%) of the 35 cases had severe intraocular inflammation, and the inflammation had some characteristics of toxic anterior segment syndrome (TASS). Five of the 5 cases had a predominantly anterior chamber reaction, and 4 of the 5 cases were accompanied by hypopyon. Undiluted samples collected from both the aqueous and vitreous of the 5 cases were culture negative. The BCVA was 0.66+/-0.29 (mean+/-standard deviations) logarithm of the minimum angle resolution (logMAR) units, and the endothelial cell density was 2683.6+/-97.3/mm(2) before the intravitreal bevacizumab. At the final visit, the BCVA was 0.44+/-0.36 logMAR units, and the cell density was 2679.0+/-217.5/mm(2). These differences were not significant (P = 0.171 and 0.964). CONCLUSIONS These observations indicate that an intravitreal injection of bevacizumab can induce sterile endophthalmitis that has characteristics of TASS.

Analysis of Transcriptional Regulatory Pathways of Photoreceptor Genes by Expression Profiling of the Otx2-Deficient Retina

In the vertebrate retina, the Otx2 transcription factor plays a crucial role in the cell fate determination of both rod and cone photoreceptors. We previously reported that Otx2 conditional knockout (CKO) mice exhibited a total absence of rods and cones in the retina due to their cell fate conversion to amacrine-like cells. In order to investigate the entire transcriptome of the Otx2 CKO retina, we compared expression profile of Otx2 CKO and wild-type retinas at P1 and P12 using microarray. We observed that expression of 101- and 1049-probe sets significantly decreased in the Otx2 CKO retina at P1 and P12, respectively, whereas, expression of 3- and 4149-probe sets increased at P1 and P12, respectively. We found that expression of genes encoding transcription factors involved in photoreceptor development, including Crx, Nrl, Nr2e3, Esrrb, and NeuroD, was markedly down-regulated in the Otx2 CKO at both P1 and P12. Furthermore, we identified three human retinal disease loci mapped in close proximity to certain down-regulated genes in the Otx2 CKO retina including Ccdc126, Tnfsf13 and Pitpnm1, suggesting that these genes are possibly responsible for these diseases. These transcriptome data sets of the Otx2 CKO retina provide a resource on developing rods and cones to further understand the molecular mechanisms underlying photoreceptor development, function and disease.

Post-translational Maturation of Dystroglycan Is Necessary for Pikachurin Binding and Ribbon Synaptic Localization

Pikachurin, the most recently identified ligand of dystroglycan, plays a crucial role in the formation of the photoreceptor ribbon synapse. It is known that glycosylation of dystroglycan is necessary for its ligand binding activity, and hypoglycosylation is associated with a group of muscular dystrophies that often involve eye abnormalities. Because little is known about the interaction between pikachurin and dystroglycan and its impact on molecular pathogenesis, here we characterize the interaction using deletion constructs and mouse models of muscular dystrophies with glycosylation defects (Largemyd and POMGnT1-deficient mice). Pikachurin-dystroglycan binding is calcium-dependent and relatively less sensitive to inhibition by heparin and high NaCl concentration, as compared with other dystroglycan ligand proteins. Using deletion constructs of the laminin globular domains in the pikachurin C terminus, we show that a certain steric structure formed by the second and the third laminin globular domains is necessary for the pikachurin-dystroglycan interaction. Binding assays using dystroglycan deletion constructs and tissue samples from Large-deficient (Largemyd) mice show that Large-dependent modification of dystroglycan is necessary for pikachurin binding. In addition, the ability of pikachurin to bind to dystroglycan prepared from POMGnT1-deficient mice is severely reduced, suggesting that modification of the GlcNAc-β1,2-branch on O-mannose is also necessary for the interaction. Immunofluorescence analysis reveals a disruption of pikachurin localization in the photoreceptor ribbon synapse of these model animals. Together, our data demonstrate that post-translational modification on O-mannose, which is mediated by Large and POMGnT1, is essential for pikachurin binding and proper localization, and suggest that their disruption underlies the molecular pathogenesis of eye abnormalities in a group of muscular dystrophies.

Heparan Sulfate Regulates Intraretinal Axon Pathfinding by Retinal Ganglion Cells

PURPOSE. Heparan sulfate (HS) is abundantly expressed in the developing neural retina; however, its role in the intraretinal axon guidance of retinal ganglion cells (RGCs) remains unclear. In this study, the authors examined whether HS was essential for the axon guidance of RGCs toward the optic nerve head. METHODS. The authors conditionally ablated the gene encoding the exostosin-1 (Ext1) enzyme, using the dickkopf homolog 3 (Dkk3)-Cre transgene, which disrupted HS expression in the mouse retina during directed pathfinding by RGC axons toward the optic nerve head. In situ hybridization, immunohistochemistry, DiI tracing, binding assay, and retinal explant assays were performed to evaluate the phenotypes of the mutants and the roles of HS in intraretinal axon guidance. RESULTS. Despite no gross abnormality in RGC distribution, the mutant RGC axons exhibited severe intraretinal guidance errors, including optic nerve hypoplasia, ectopic axon penetration through the full thickness of the neural retina and into the subretinal space, and disturbance of the centrifugal projection of RGC axons toward the optic nerve head. These abnormal phenotypes shared similarities with the RGC axon misguidance caused by mutations of genes encoding Netrin-1 and Slit-1/2. Explant assays revealed that the mutant RGCs exhibited disturbed Netrin-1-dependent axon outgrowth and Slit-2-dependent repulsion. CONCLUSIONS. The present study demonstrated that RGC axon projection toward the optic nerve head requires the expression of HS in the neural retina, suggesting that HS in the retina functions as an essential modulator of Netrin-1 and Slit-mediated intraretinal RGC axon guidance.

Prediction of letter contrast sensitivity using videokeratographic indices

PURPOSE To analyze the relationship between corneal topography and letter contrast sensitivity. METHOD Experiments were conducted on 59 eyes of 51 patients who had best spectacle-corrected visual acuity of 20/20 or better and no ocular pathology except for the corneal shape. Thirty-nine eyes had an abnormal topographic pattern resulting from keratoconus, and the other 20 eyes showed a normal topographic pattern. Videokeratography was performed with the TMS-2 videokeratoscope, and the surface regularity index, surface asymmetry index, and coefficient of variation of power were obtained for each subject. Letter contrast sensitivity was measured with the CSV-1000LV with spectacle correction. The correlation between the number of correct letters and topographic indices was calculated. RESULTS The abnormal topography group had a significantly greater loss of letter contrast sensitivity (median = 20 letters) than the normal control (median = 23 letters; P =.0001). There were statistically significant correlations between number of correct letters and the coefficient of variation of power (r = -.77; P =. 001), number of correct letters and surface regularity index (r = -. 76, P =.001), and the number of correct letters and surface asymmetry index (r = -.64; P =.001). The linear regression equation between number of correct letters and the coefficient of variation of power was the number of correct letters = -0.05 x the coefficient of variation of power + 23.2. CONCLUSIONS Our results suggest that subtle visual deteriorations, which are barely detected by contrast sensitivity testing, can be predicted objectively by the corneal topographic indices.

Analysis of Vitrectomy for Idiopathic Macular Hole by Optical Coherence Tomography

PURPOSE To evaluate foveal structure after vitrectomy for idiopathic macular hole in relation to postoperative visual outcome. METHODS Optical coherence tomography was performed postoperatively to assess retinal thickness at the foveal center in patients who underwent vitrectomy, posterior hyaloid membrane removal, and perfluoropropane gas tamponade for idiopathic macular hole. Thirty-seven eyes of 36 patients documented to have achieved anatomic hole closure by optical coherence tomography were included in the study. RESULTS Increased visual acuity significantly correlated with greater foveal thickness assessed at a median of 5 months postoperatively (Spearman analysis; R = .453, P = .005). CONCLUSION Visual outcome after anatomic closure of macular holes by vitrectomy is closely related to the structure of the center of the fovea postoperatively.

Multiple iridociliary cysts in patients with mucopolysaccharidoses

The mucopolysaccharidoses (MPSs) are rare hereditary diseases. They are classified into six types by the distinct lysosomal accumulations of glycosaminoglycans, which give rise to the progressive clinical features with involvement of multisystems. Ophthalmic complications, such as corneal stromal opacity, pigmentary retinal degeneration, optic nerve atrophy, and glaucoma, are common in patients with MPSs. Cysts in various organs have been reported in patients with MPSs—for example, multiple dentigerous cysts, multifocal large cysts in the white matter and arachnoid of the brain, and bone cysts.1,2 In the eye, membrane bound vacuoles in the non-pigmented epithelium of the ciliary processes have been observed by electron microscopy.3 However, iridociliary cysts have never been reported in patients with MPSs. We present two cases of multiple iridociliary cysts in two patients with MPSs, one with Scheie syndrome and the other with Maroteaux-Lamy syndrome. ### Case 1 A 18 year old woman, who was diagnosed with Scheie syndrome (MPS type IS) by enzyme assay. The activity of α-l-iduronidase in peripheral blood lymphocytes was …