The retinal degeneration slow (rds) gene product is a photoreceptor disc membrane-associated glycoprotein

Mice homozygous for the retinal degeneration slow (rds) mutation exhibit abnormal development of photoreceptor cells, followed by their slow degeneration. We have recently cloned the rds gene and determined the structure of the wild-type rds mRNA. Here we show that the gene is expressed exclusively in photoreceptor cells. We demonstrate that it encodes a 39 kd membrane-associated glycoprotein that is restricted to photoreceptor outer segments. By electron microscopy, we show that the rds protein is distributed uniformly within outer segment discs. The developmental appearance of the rds protein coincides with outer segment disc formation. We propose that the rds protein functions as an adhesion molecule for stabilization of the outer segment discs.     

Oxygen-induced retinopathy in mice with retinal photoreceptor cell degeneration

Aims

It is reported that retinal neovascularization seems to rarely co-exist with retinitis pigmentosa in patients and in some mouse models; however, it is not widely acknowledged as a universal phenomenon in all strains of all animal species. We aimed to further explore this phenomenon with an oxygen-induced retinopathy model in mice with retinal photoreceptor cell degeneration.

Main methods

Oxygen-induced retinopathy of colored and albino mice with rapid retinal degeneration were compared to homologous wild-type mice. The retinas were analyzed using high-molecular-weight FITC-dextran stained flat-mount preparation, hematoxylin and eosin (H&E) stained cross-sections, an immunohistochemical test for vascular endothelial growth factor (VEGF) distribution and Western blotting for VEGF expression after exposure to hyperoxia between postnatal days 17 (P17) and 21.

Key findings

Leakage and areas of non-perfusion of the retinal blood vessels were alleviated in the retinal degeneration mice. The number of preretinal vascular endothelial cell nuclei in the retinal degeneration mice was smaller than that in the homologous wild-type mice after exposure to hyperoxia (P < 0.01). The degree of oxygen-induced retinopathy was positively correlated with the VEGF expression level. However, the VEGF expression level was lower in the retinal degeneration mice.

Significance

Proliferative retinopathy occurred in mice with rapid retinal degeneration, but retinal photoreceptor cell degeneration could partially restrain the retinal neovascularization in this rapid retinal degeneration mouse model.     

Retinal degeneration-3 protein attenuates photoreceptor degeneration in transgenic mice expressing dominant mutation of human retinal guanylyl cyclase

Different forms of photoreceptor degeneration cause blindness. Retinal degeneration-3 protein (RD3) deficiency in photoreceptors leads to recessive congenital blindness. We proposed that aberrant activation of the retinal membrane guanylyl cyclase (RetGC) by its calcium-sensor proteins (guanylyl cyclase–activating protein [GCAP]) causes this retinal degeneration and that RD3 protects photoreceptors by preventing such activation. We here present in vivo evidence that RD3 protects photoreceptors by suppressing activation of both RetGC1 and RetGC2 isozymes. We further suggested that insufficient inhibition of RetGC by RD3 could contribute to some dominant forms of retinal degeneration. The R838S substitution in RetGC1 that causes autosomal-dominant cone–rod dystrophy 6, not only impedes deceleration of RetGC1 activity by Ca²⁺GCAPs but also elevates this isozyme''s resistance to inhibition by RD3. We found that RD3 prolongs the survival of photoreceptors in transgenic mice harboring human R838S RetGC1 (R838S⁺). Overexpression of GFP-tagged human RD3 did not improve the calcium sensitivity of cGMP production in R838S⁺ retinas but slowed the progression of retinal blindness and photoreceptor degeneration. Fluorescence of the GFP-tagged RD3 in the retina only partially overlapped with immunofluorescence of RetGC1 or GCAP1, indicating that RD3 separates from the enzyme before the RetGC1:GCAP1 complex is formed in the photoreceptor outer segment. Most importantly, our in vivo results indicate that, in addition to the abnormal Ca²⁺ sensitivity of R838S RetGC1 in the outer segment, the mutated RetGC1 becomes resistant to inhibition by RD3 in a different cellular compartment(s) and suggest that RD3 overexpression could be utilized to reduce the severity of cone–rod dystrophy 6 pathology.     

Iron-mediated retinal degeneration in haemojuvelin-knockout mice

Haemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE (human leucocyte antigen-like protein involved in iron homoeostasis), transferrin receptor 2, ferroportin, hepcidin and HJV (haemojuvelin). Recent studies have established the expression of all of the five genes in the retina, indicating their importance in retinal iron homoeostasis. Previously, we demonstrated that HJV is expressed in RPE (retinal pigment epithelium), the outer and inner nuclear layers and the ganglion cell layer. In the present paper, we report on the consequences of Hjv deletion on the retina in mice. Hjv-/- mice at ≥18 months of age had increased iron accumulation in the retina with marked morphological damage compared with age-matched controls; these changes were not found in younger mice. The retinal phenotype in Hjv-/- mice included hyperplasia of RPE. We isolated RPE cells from wild-type and Hjv-/- mice and examined their growth patterns. Hjv-/- RPE cells were less senescent and exhibited a hyperproliferative phenotype. Hjv-/- RPE cells also showed up-regulation of Slc7a11 (solute carrier family 7 member 11 gene), which encodes the 'transporter proper' subunit xCT in the heterodimeric amino acid transporter xCT/4F2hc (cystine/glutamate exchanger). BMP6 (bone morphogenetic protein 6) could not induce hepcidin expression in Hjv-/- RPE cells, confirming that retinal cells require HJV for induction of hepcidin via BMP6 signalling. HJV is a glycosylphosphatidylinositol-anchored protein, and the membrane-associated HJV is necessary for BMP6-mediated activation of hepcidin promoter in RPE cells. Taken together, these results confirm the biological importance of HJV in the regulation of iron homoeostasis in the retina and in RPE.     

Upregulation of vascular endothelial growth factor (VEGF) in the retinas of transgenic mice overexpressing interleukin-1beta (IL-1beta) in the lens and mice undergoing retinal degeneration

IL-1beta is a pro-inflammatory agent associated with angiogenesis and increased vascular permeability. To determine whether IL-1beta elicits these responses through an upregulation of VEGF, transgenic mice that overexpress IL-1beta in the lens were evaluated at various time points for the localization of VEGF, the location and extent of blood-retinal barrier (BRB) breakdown, and the origin and extent of neovascularization (NV). In homozygous and heterozygous transgenic mice, but not controls, intense VEGF immunoreactivity was scattered throughout the retina at postnatal days 5-7 (P5-7), just after the onset of inflammatory cell infiltration. VEGF staining in the retina remained widespread, but weak from P9-15. Beginning at P15, the intensity of VEGF immunoreactivity achieved a second peak, which it maintained through adulthood. This peak coincided with significant retinal destruction due to massive inflammation. The onset of BRB breakdown coincided with the upregulation of VEGF (P5-7) and widespread BRB breakdown was demonstrated from about P9. From P9-12, aggregates of cells positive for Griffonia simplicifolia isolectin-B4, a marker for vascular endothelial cells, formed on the retinal surface. These cells migrated into the retina at P12-15 with the more superficial cells forming a network of vessels and the deeper cells remaining in small clusters, thus demonstrating that NV occurs much later than BRB breakdown. Non-transgenic FVB/N mice, which undergo retinal degeneration beginning at about P9, also demonstrate the latter peak of VEGF upregulation and the accompanying BRB breakdown, but not the early upregulation. VEGF immunostaining of transgenic and non-transgenic mouse retinas was eliminated by pre-incubation of the VEGF antibodies with VEGF peptide. The data suggest that the early peak of VEGF upregulation (P5-7) and its accompanying BRB breakdown is due to IL-1beta expression and is likely to be dependent on inflammatory cell infiltration. The latter peak appears to be related to retinal destruction.     

Lack of causal relationship between clusterin expression and photoreceptor apoptosis in light-induced retinal degeneration

Induction of apoptosis in the retina leads to cellular death by molecular mechanisms that are not well understood. Clusterin expression is increased in tissues undergoing apoptosis, including retinal neurodegenerative states, but the causal relationships remain to be clarified. To gain insight into clusterin's role in photoreceptor apoptosis, the cellular distribution of clusterin mRNA was compared with the pattern of apoptotic nuclear labelling in a rat model of light-induced retinal degeneration. In control retinal sections, clusterin mRNA was localized to the retinal pigment epithelium cells, photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. Clusterin expression decreased in photoreceptors and retinal pigment epithelium cells, which progressively degenerated, and increased in preserved inner nuclear layer, in proportion to the duration of light exposure in both cyclic light- and dark-reared animals. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor death, but may relate to cytoprotective functions.     

Yttrium oxide nanoparticles prevent photoreceptor death in a light-damage model of retinal degeneration

Photoreceptor (PR) cells are prone to accumulation of reactive oxygen species (ROS) and oxidative stress. An imbalance between the production of ROS and cellular antioxidant defenses contributes to PR degeneration and blindness in many different ocular disease states. Yttrium oxide (Y₂O₃) nanoparticles (NPs) are excellent free radical scavengers owing to their nonstoichiometric crystal defects. Here we utilize a murine light-stress model to test the efficacy of Y₂O₃ NPs (~10–14 nm in diameter) in ameliorating retinal oxidative stress-associated degeneration. Our studies demonstrate that intravitreal injections of these NPs at doses ranging from 0.1 to 5.0 µM 2 weeks before acute light stress protect PRs from degeneration. This protection is reflected both structurally (i.e., decreased light-associated thinning of the outer nuclear layer) and functionally (i.e., preservation of scotopic and photopic electroretinogram amplitudes). We also observe preservation of structure and function when NPs are delivered immediately after acute light stress, although the magnitude of the preservation is smaller, and only doses ranging from 1.0 to 5.0 µM were effective. We show that the Y₂O₃ NPs are nontoxic and well tolerated after intravitreal delivery. Our results suggest that Y₂O₃ NPs have astonishing antioxidant benefits and, with further exploration, may be an excellent strategy for the treatment of oxidative stress associated with multiple forms of retinal degeneration.     

Assessment of retinal degeneration in outbred albino mice

Evaluation of a pharmaceutical's safety includes assessment of the potential for ophthalmologic toxicity. These nonclinical studies commonly use various outbred stocks of mice. Pretest indirect ophthalmoscopic examinations in the commonly used outbred stock Hsd:ICR(CD-1) indicated that retinal degeneration was a problem in this particular outbred stock of mice. This prompted the authors to examine other stocks of outbred mice routinely used in the performance of nonclinical safety studies. Groups of mice were observed over a 13-week period to determine the progression and changing incidence of retinal degeneration. Light intensity in the room and caging was measured during the study, and it was determined that light did not play a direct role in the progression of the retinal degeneration observed during the study. Histomorphologic examination of the mouse eyes was performed at the end of the study to confirm the presence of retinal degeneration observed after ophthalmoscopic examination. The incidence of retinal atrophy in the various outbred stocks of mice was: Crl:CFW(SW)BR (98.3%), Tac(SW)fBR (80%), Tac:Icr:Ha(ICR)fBR (75%), Hsd:ICR(CD-1) (43.3%), and Crl:CF-1BR (3.0%). Retinal atrophy was not observed in the following outbred mice stocks: Crl:CD-1(ICR)BR, HsdWin:CFW1, and Hsd:NSA(CF-1). On the basis of these findings, it is highly recommended that pretest ophthalmologic screening be performed on mice to obviate pre-existing conditions from confounding or invalidating nonclinical study results.     

Modification of glial-neuronal cell interactions prevents photoreceptor apoptosis during light-induced retinal degeneration

Prolonged or high-intensity exposure to visible light leads to photoreceptor cell death. In this study, we demonstrate a novel pathway of light-induced photoreceptor apoptosis involving the low-affinity neurotrophin receptor p75 (p75NTR). Retinal degeneration upregulated both p75NTR and the high-affinity neurotrophin receptor TrkC in different parts of Müller glial cells. Exogenous neurotrophin-3 (NT-3) increased, but nerve growth factor (NGF) decreased basic fibroblast growth factor (bFGF) production in Müller cells, which can directly rescue photoreceptor apoptosis. Blockade of p75NTR prevented bFGF reduction and resulted in both structural and functional photoreceptor survival in vivo. Furthermore, the absence of p75NTR significantly prevented light-induced photoreceptor apoptosis. These observations implicate glial cells in the determination of neural cell survival, and suggest functional glial-neuronal cell interactions as new therapeutic targets for neurodegeneration.     

Colorectal hyperplasia and dysplasia due to human carcinoembryonic antigen (CEA) family member expression in transgenic mice

CEA and CEACAM6 are immunoglobulin family intercellular adhesion molecules that are up-regulated without structural mutations in approximately 70% of human cancers. Results in in vitro systems showing tumorigenic effects for these molecules suggest that this correlation could indicate an instrumental role in tumorigenesis. To test whether this applies in vivo, transgenic mice harboring 187 kb of the human genome containing four CEA family member genes including the CEA and CEACAM6 genes were created and their copy numbers increased by mating until colonocyte expression levels reached levels seen in human colorectal carcinomas. The colonocyte surface level of integrin alpha5 and the activation of AKT increased progressively with the expression levels of CEA/CEACAM6. Colonic crypts showed a progressive increase in colonocyte proliferation, an increase in crypt fission, and a strong inhibition of both differentiation and anoikis/apoptosis. All transgenic mice showed massively enlarged colons comprising a continuous mosaic of severe hyperplasia, dysplasia and serrated adenomatous morphology. These results suggest that up-regulated non-mutated adhesion molecules could have a significant instrumental role in human cancer.     

Abnormal photoreceptor outer segment development and early retinal degeneration in kif3a mutant zebrafish

Photoreceptors are highly specialized sensory neurons that possess a modified primary cilium called the outer segment. Photoreceptor outer segment formation and maintenance require highly active protein transport via a process known as intraflagellar transport. Anterograde transport in outer segments is powered by the heterotrimeric kinesin II and coordinated by intraflagellar transport proteins. Here, we describe a new zebrafish model carrying a nonsense mutation in the kinesin II family member 3A (kif3a) gene. Kif3a mutant zebrafish exhibited curved body axes and kidney cysts. Outer segments were not formed in most parts of the mutant retina, and rhodopsin was mislocalized, suggesting KIF3A has a role in rhodopsin trafficking. Both rod and cone photoreceptors degenerated rapidly between 4 and 9 days post fertilization, and electroretinography response was not detected in 7 days post fertilization mutant larvae. Loss of KIF3A in zebrafish also resulted in an intracellular transport defect affecting anterograde but not retrograde transport of organelles. Our results indicate KIF3A plays a conserved role in photoreceptor outer segment formation and intracellular transport.     

Rescue of photoreceptor degeneration by curcumin in transgenic rats with P23H rhodopsin mutation

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects.     

转人APP基因小鼠视网膜视神经细胞超微结构改变

目的观察转人APP基因AD模型小鼠视网膜各层细胞超微结构的改变。方法实验采用C57BL/6J转人APP基因小鼠6只,采用同背景10个月龄的C57 BL/6J正常小鼠6只做为对照。灌注处死小鼠,完整取出右眼球分离视网膜,制作电镜标本观察视网膜各层细胞超微结构改变。结果与对照组相比,模型组视网膜各层细胞异常明显:膜盘排列结构模糊,局部间隙溶解、甚至消失;外核层细胞体干枯变形,染色质聚集浓缩;内核层可见固缩细胞;神经节细胞胞膜不完整,线粒体水肿,并可见染色质聚集。结论转人APP基因AD模型小鼠视网膜各层神经细胞超微结构存在明显病理改变。     

Complete RNAi rescue of neuronal degeneration in a constitutively active Drosophila TRP channel mutant

RNA interference has been widely used to reduce the quantity of the proteins encoded by the targeted genes. A constitutively active, dominant allele of trp, TrpP365, causes massive degeneration of photoreceptors through a persistent and excessive Ca2+ influx. Here we show that a substantial reduction of the TRP channel protein by RNAi in TrpP365 heterozygotes completely rescues the neuronal degeneration and significantly improves the light-elicited responses of the eye. The reduction need not be complete, suggesting that rescue of degeneration may be possible with minimal side effects arising from overdepletion of the target protein.     

A simple polymerase chain reaction assay for genotyping the retinal degeneration mutation (Pdeb(rd1)) in FVB/N-derived transgenic mice

FVB/N mice are one of the most common inbred strains for the generation of transgenic animals. This mouse strain is preferred for transgenesis because of its fertilized oocytes, which have unique pronuclei for microinjection, and its vigorous reproductive performance along with consistently large litter sizes. However, these inbred mice carry a retinal degeneration mutation caused by a proviral insertion into the Pdeb gene, encoding the beta subunit of cGMP phosphodiesterase. This mutation (Pdeb(rd1), formerly known as rd) results in postnatal rod photoreceptor degeneration and causes severe visual impairment, which may be relevant for behavioural and vision-related research. This deficit can be overcome by crossing these mice with other mouse strains carrying the wild-type allele at the Pdeb locus. We have devised a simple polymerase chain reaction (PCR)-based method for distinguishing between the mutant and the wild-type alleles, thus allowing the efficient monitoring of the Pdeb(rd1) mutation in FVB/N-derived transgenic mice prior to experimentation where visual deficit is expected to have an influence in the phenotype.