Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

Background Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains. Results We examined the localization of Crb2a constructs and their effects on rod morphology. We found that only the full-length Crb2a protein approximated the normal localization of Crb2a protein apical to adherens junctions in the photoreceptor inner segment. Several Crb2a construct proteins localized abnormally to the outer segment and one construct localized abnormally to the cell body. Overexpression of full-length Crb2a greatly increased inner segment size while expression of several other constructs increased outer segment size. Conclusions Our observations suggest that particular domains in Crb2a regulate its localization and thus may regulate its regionalized function. Our results also suggest that the PDZ-binding domain in Crb2a might bring a protein(s) into the Crb complex that alters the function of the FERM-binding domain

Defects in the Outer Limiting Membrane Are Associated with Rosette Development in the Nrl−/− Retina

The neural retinal leucine zipper (Nrl) knockout mouse is a widely used model to study cone photoreceptor development, physiology, and molecular biology in the absence of rods. In the Nrl−/− retina, rods are converted into functional cone-like cells. The Nrl−/− retina is characterized by large undulations of the outer nuclear layer (ONL) commonly known as rosettes. Here we explore the mechanism of rosette development in the Nrl−/− retina. We report that rosettes first appear at postnatal day (P)8, and that the structure of nascent rosettes is morphologically distinct from what is seen in the adult retina. The lumen of these nascent rosettes contains a population of aberrant cells protruding into the subretinal space that induce infolding of the ONL. Morphologically adult rosettes do not contain any cell bodies and are first detected at P15. The cells found in nascent rosettes are photoreceptors in origin but lack inner and outer segments. We show that the adherens junctions between photoreceptors and Müller glia which comprise the retinal outer limiting membrane (OLM) are not uniformly formed in the Nrl−/− retina and thus allow protrusion of a population of developing photoreceptors into the subretinal space where their maturation becomes delayed. These data suggest that the rosettes of the Nrl−/− retina arise due to defects in the OLM and delayed maturation of a subset of photoreceptors, and that rods may play an important role in the proper formation of the OLM

Anti-inflammatory recombinant TSG-6 stabilizes the progression of focal retinal degeneration in a murine model

<p>Abstract</p> <p>Background</p> <p>Inflammatory responses are detected in the retina of patients with age-related macular degeneration and <it>Ccl2^-/-/Cx3cr1^-/-</it>mice on rd8 background,(<it>Ccl2^-/-/Cx3cr1^-/-</it>mice) a model that develops progressive age-related macular degeneration-like retinal lesions including focal photoreceptor degeneration, abnormal retinal pigment epithelium and A2E accumulation. Tumor necrosis factor-inducible gene 6 protein is an anti-inflammatory protein and has been shown to improve myocardial infarction outcome and chemically injured cornea in mice by suppressing inflammation. In this study, we evaluated the effect of an intravitreous injection of recombinant TSG-6 on the retinal lesions of <it>Ccl2^-/-/Cx3cr1^-/-</it>mice.</p> <p>Methods</p> <p>Recombinant TSG-6 (400 ng) was administered by intravitreous injection into the right eye of six-week-old C<it>cl2^-/-/Cx3cr1^-/-</it>mice. Their left eye was injected with phosphate-buffered saline as a control. Funduscopic pictures were taken before injection and sequentially once a month after injection. The mice were killed two months after injection and the ocular histology examined. Retinal A2E, a major component of lipofuscin, was measured by high performance liquid chromatography. The microarray of ocular mRNA of 92 immunological genes was performed. The genes showing differentiated expression in microarray were further compared between the injected right eye and the contralateral (control) eye by [real-time quantitative reverse transcription polymerase chain reaction] qRT-PCR.</p> <p>Results</p> <p>The continuous monitoring of the fundus for two months showed a slower progression or alleviation of retinal lesions in the treated right eyes as compared with the untreated left eyes. Among 23 pairs of eyes, the lesion levels improved in 78.3%, stayed the same in 8.7% and progressed in 13.0%. Histology confirmed the clinical observation. Even though there was no difference in the level of A2E between the treated and the untreated eyes, microarray analysis of 92 immune genes showed that <it>IL-17a </it>was substantially decreased after the treatment. Expression of <it>TNF-α </it>showed a similar pattern to <it>IL-17a</it>. The results were consistent in duplicated arrays and confirmed by qRT-PCR.</p> <p>Conclusions</p> <p>We concluded that intravitreous administration of recombinant TSG-6 might stabilize retinal lesions in <it>Ccl2^-/-/Cx3cr1^-/-</it>mice on rd8 background. Modulation of ocular immunological gene expressions, especially IL-17a, could be one of the mechanisms.</p

GFAP-Driven GFP Expression in Activated Mouse Muller Glial Cells Aligning Retinal Blood Vessels Following Intravitreal Injection of AAV2/6 Vectors

Background: Muller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Muller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.Methodology/Principal Findings: We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Muller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Muller glial cells, several other inner retinal cell types were transduced. To obtain Muller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1(-/-) retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Muller glial cells aligning retinal blood vessels.Conclusions/Significance: Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells

A clinical and molecular characterisation of CRB1-associated maculopathy

To date, over 150 disease-associated variants in CRB1 have been described, resulting in a range of retinal disease phenotypes including Leber congenital amaurosis and retinitis pigmentosa. Despite this, no genotype–phenotype correlations are currently recognised. We performed a retrospective review of electronic patient records to identify patients with macular dystrophy due to bi-allelic variants in CRB1. In total, seven unrelated individuals were identified. The median age at presentation was 21 years, with a median acuity of 0.55 decimalised Snellen units (IQR = 0.43). The follow-up period ranged from 0 to 19 years (median = 2.0 years), with a median final decimalised Snellen acuity of 0.65 (IQR = 0.70). Fundoscopy revealed only a subtly altered foveal reflex, which evolved into a bull’s-eye pattern of outer retinal atrophy. Optical coherence tomography identified structural changes—intraretinal cysts in the early stages of disease, and later outer retinal atrophy. Genetic testing revealed that one rare allele (c.498_506del, p.(Ile167_Gly169del)) was present in all patients, with one patient being homozygous for the variant and six being heterozygous. In trans with this, one variant recurred twice (p.(Cys896Ter)), while the four remaining alleles were each observed once (p.(Pro1381Thr), p.(Ser478ProfsTer24), p.(Cys195Phe) and p.(Arg764Cys)). These findings show that the rare CRB1 variant, c.498_506del, is strongly associated with localised retinal dysfunction. The clinical findings are much milder than those observed with bi-allelic, loss-of-function variants in CRB1, suggesting this in-frame deletion acts as a hypomorphic allele. This is the most prevalent disease-causing CRB1 variant identified in the non-Asian population to date

ERG Alteration Due to the rd8 Mutation of the Crb1 Gene in Cln3 (+/+) (rd8-/rd8-) Mice

Mattapallil et al. described that vendor lines for C57BL/6 N mice may carry the rd8 mutation that leads to an ocular phenotype, which could be mistaken for an induced retinal degeneration. This mouse strain is widely used in ophthalmic research as a background for modeling retinal degeneration. In the process of studying Cln3(Delta ex7/8) knock-in mice on a C57BL/6 N background, we became aware of this issue. The aim of this study thus was to use electroretinography to investigate the age-dependent functional loss in Cln3(+/+) (rd8-/rd8-) mice and compare it to C57BL/6 J mice. The scotopic and photopic amplitudes of the a-wave and b-wave decrease significantly in mutant mice with increasing age, and the implicit time is prolonged. Especially the oscillatory potentials arising from inner retinal interaction seem to be notably affected by the rd8 mutation. Surprisingly, the amplitudes in young C57BL/6 J mice were lower than those measured in C57BL/6 N at any time point. Our results indicate that the rd8 mutation present in C57BL/6 N mice affects the function of the inner and outer retina. This is surprising given that the major retinal morphological alterations due to the rd8 mutation are found in the outer retina. We conclude that the rd8 mutation does affect the retinal function in Cln3(+/+ rd8-/rd8-) mice in a variable manner. Epigenetic factors and modifying genes lead to a phenotype shift in these mice. Interpreting the results of previous studies in mutant mice on C57BL/6 N background is challenging as comparing results obtained in independent studies or on other mouse backgrounds may be misleading. Using littermates as controls remains the only valid option

The Impact of Adherens and Tight Junctions on Physiological Function and Pathological Changes in the Retina.

The formation of solid tissues is not a simple aggregation of individual cells but rather an ordered assembly of cells connected by junctions. These junctions provide a diffusion barrier as well as mechanical support and a conduit for signalling changes in the environment to the cells. Cell junctions are functionally categorized as occluding (e.g. tight junctions, TJs), anchoring (e.g. adherens junctions, AJs) and communicating junctions (e.g. gap junctions). Each type of the cell junction is formed by protein complexes with extracellular domains and/or intracellular domains, which bind partners that provide scaffolding and signalling components. Cell junctions are ubiquitously expressed in multiple tissues and organs, including the retina. In the retina, their biological impact is not limited to regulating tissue growth and development. Disruption of the complexes mediates both congenital and postnatal pathogenesis. In this review, we will focus on cell junctions, specifically AJs and TJs in the external limiting membrane, in order to articulate their influence on pathophysiology of the retina. Adv Exp Med Biol 2018; 1074:545-551