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Primary cilia are conserved organelles found in polarized cells within the eye that regulate cell growth, migration, and differentiation. Although the role of cilia in photoreceptors is well-studied, the formation of cilia in other retinal cell types has received little attention. In this study, we examined the ciliary profile focused on the inner nuclear layer of retinas in mice and rhesus macaque primates. METHODS.Retinal sections or flatmounts from Arl13b-Cetn2 tg transgenic mice were immunostained for cell markers (Pax6, Sox9, Chx10, Calbindin, Calretinin, ChaT, GAD67, Prox1, TH, and vGluT3) and analyzed by confocal microscopy. Primate retinal sections were immunostained for ciliary and cell markers (Pax6 and Arl13b). Optical coherence tomography (OCT) and ERGs were used to assess visual function of Vift88 mice. RESULTS.During different stages of mouse postnatal eye development, we found that cilia are present in Pax6-positive amacrine cells, which were also observed in primate retinas. The cilia of subtypes of amacrine cells in mice were shown by immunostaining and electron microscopy. We also removed primary cilia from vGluT3 amacrine cells in mouse and found no significant vision defects. In addition, cilia were present in the outer limiting membrane, suggesting that a population of Müller glial cells forms cilia. CONCLUSIONS.We report that several subpopulations of amacrine cells in inner nuclear layers of the retina form cilia during early retinal development in mice and primates.
Steroid-induced glaucoma is a common form of secondary open angle glaucoma characterized by ocular hypertension (elevated intraocular pressure [IOP]) in response to prolonged glucocorticoid exposure. Elevated IOP occurs with increased outflow resistance and altered trabecular meshwork (TM) function. Recently, we used an optogenetic approach in TM to regulate the 5-phosphatase, OCRL, which contributes to regulating PI(4,5)P2 levels. Here, we applied this system with the aim of reversing compromised outflow function in a steroid-induced ocular hypertension mouse model. Methods: Elevated IOP was induced by chronic subconjunctival dexamethasone injections in wild-type C57Bl/6j mice. AAV2 viruses containing optogenetic modules of cryptochrome 2 (Cry2)-OCRL-5ptase and CIBN-GFP were injected into the anterior chamber. Four weeks after viral expression and dexamethasone exposure, IOP was measured by tonometer and outflow facility was measured by perfusion apparatus. Human TM cells were treated with dexamethasone, stimulated by light and treated with rhodamine-phalloidin to analyze actin structure.Results: Dexamethasone treatment elevated IOP and decreased outflow facility in wildtype mice. Optogenetic constructs were expressed in the TM of mouse eyes. Light stimulation caused CRY2-OCRL-5ptase to translocate to plasma membrane (CIBN-CAAX-GFP) and cilia (CIBN-SSTR3-GFP) in TM cells, which rescued the IOP and outflow facility. In addition, aberrant actin structures formed by dexamethasone treatment were reduced by optogenetic stimulation in human TM cells in culture.Conclusions: Subcellular targeting of inositol phosphatases to remove PIP2 represents a promising strategy to reverse defective TM function in steroid-induced ocular hypertension.
Senior-Løken syndrome is a rare genetic disorder which presents with nephronophthisis and retinal degeneration, leading to end-stage renal disease and progressive blindness. The most frequent cause of juvenile nephronophthisis is mutation in the nephronophthisis type 1 (NPHP1) gene. NPHP1 encodes the protein nephrocystin-1, which functions at the transition zone (TZ) of primary cilia. Here we report a 9-year-old Senior-Løken syndrome patient with NPHP1 deletion, who presents with a decreased electroretinogram consistent with early retinal degeneration. The patient had undergone bilateral nephrectomy and a renal transplant. Immunohistochemistry and electron microscopy of the resected kidney showed disorganized cystic structures with loss of cilia in renal tubules. Phosphoinositides have been recently recognized as critical components of the ciliary membrane and immunostaining of kidney sections for phosphoinositide 5-phosphatases (INPP5E) showed loss of staining compared to a healthy control. The decreased expression of INPP5E specifically in the primary cilium, coupled with disorganized cilia morphology, suggests a novel role of NPHP1 that it is involved in regulating ciliary phosphoinositide in the ciliary membrane of renal tubular cells.
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