Isolation of a cDNA encoding a photoreceptor cell-specific actin-bundling protein: retinal fascin.

We have isolated a novel retina-specific gene, retinal fascin, encoding a new member of actin-bundling protein gene family, from a bovine retina cDNA library. The cDNA encodes a 492 amino acid protein which shows 36-57% amino acid identity with three vertebrate fascins, echinoid fascin and Drosophila singed gene. Northern blot analysis revealed that retinal fascin mRNA was exclusively expressed in the eye and not seen in other tissues examined. In situ hybridization analysis indicated that retinal fascin mRNA signals were found only in the inner segment of the photoreceptor layer and outer nuclear layer, indicating that retinal fascin was specifically expressed in photoreceptor cells. As fascins are actin-bundling proteins important for constructing several intracellular structures, retinal fascin might play a pivotal role in photoreceptor cell-specific events, such as disk morphogenesis.

Recessive mutations in the RLBP1 gene encoding cellular retinaldehyde-binding protein in a form of retinitis punctata albescens.

PURPOSE: To determine the frequency and spectrum of mutations in the RLBP1 gene encoding cellular retinaldehyde-binding protein (CRALBP) in patients with hereditary retinal degeneration. METHODS: The single-strand conformation polymorphism (SSCP) technique and a direct genomic sequencing technique were used to screen the coding exons of this gene (exons 2-8) for mutations in 324 unrelated patients with recessive or isolate retinitis pigmentosa, retinitis punctata albescens, Leber congenital amaurosis, or a related disease. Variant DNA fragments revealed by SSCP analysis were subsequently sequenced. Selected alleles that altered the coding region or intron splice sites were evaluated further through segregation analysis in the families of the index cases. RESULTS: Four novel mutations were identified in this gene among three unrelated patients with recessively inherited retinitis punctata albescens. Two of the mutations were missense: one was a frameshift, and one affected a canonical splice donor site. CONCLUSIONS: Recessive mutations in the RLBP1 gene are an uncommon cause of retinal degeneration in humans. The phenotype produced by RLBP1 mutations seems to be a form of retinitis punctata albescens.

Mutations in the 11-cis retinol dehydrogenase gene in Japanese patients with Fundus albipunctatus.

PURPOSE: To detect mutations in the RDH5 gene encoding 11-cis retinol dehydrogenase in patients from Japan with fundus albipunctatus. METHODS: Polymerase chain reaction and direct genomic sequencing techniques were used to detect mutations of the RDH5 coding exons (exons 2-5) in two unrelated patients with fundus albipunctatus. Selected alleles that altered the coding region or intron splice sites were evaluated further through segregation analysis in the families of the index cases. RESULTS: Two novel RDH5 mutations were identified. One of these was a missense mutation Val264Gly in exon 5, and the other was an in-frame insertion of 3 bp in exon 5. CONCLUSIONS: The data indicate that mutations in RDH5 are the primary cause of fundus albipunctatus.

Retinal fascin: functional nature, subcellular distribution, and chromosomal localization.

PURPOSE: To investigate the functional properties, subcellular localization, and chromosomal location of retinal fascin. METHODS: Recombinant retinal fascin protein was prepared by using a baculovirus-insect expression system. Actin-binding and -bundling assays were performed with chick actin purified from skeletal muscle. Western blot analysis and immunohistochemistry were performed with a polyclonal antibody raised against bovine retinal fascin. A human retinal cDNA library was screened with an expressed sequence tag cDNA fragment. Chromosomal location was determined with fluorescent in situ hybridization. RESULTS: The actin-binding and actin-bundling activities of retinal fascin were demonstrated by high- and low-speed centrifugation assays. Formation of filamentous (F)-actin bundles by retinal fascin in vitro was also morphologically confirmed by fluorescence microscopy and electron microscopy. Immunohistochemical analysis revealed that retinal fascin protein was localized specifically in the outer and inner segments of the photoreceptor cells in the retina. Two splicing variants of human retinal fascin cDNA were also located. One clone encoded 492 amino acids, and the other encoded 516 amino acids. The gene encoding retinal fascin was localized to human chromosome 17, region q24 -25. CONCLUSIONS: These results suggest that retinal fascin may play a role in formation of unique morphologic structures of the photoreceptor cells and is a candidate gene for retinal degenerative disorders.

Expression of Na+/myo-inositol cotransporter mRNA in the inner ear of the rat.

We have demonstrated the cellular localization of Na+/myo-inositol cotransporter (SMIT) mRNA in the rat inner ear by in situ hybridization. In the cochlea, the most intense SMIT mRNA signals were observed in fibrocytes of the spiral ligament, moderate signals were found in the spiral limbus, inner hair cells and spiral ganglion cells, while the hybridization signals were almost undetectable in the marginal cells of the stria vascularis and outer hair cells. In the vestibular system, moderate hybridization signals were found in the sensory epithelium, fibrocytes and vestibular ganglion cells. These findings suggest that SMIT plays an important role in maintenance of intracellular ionic balance and cell volume in the inner ear, especially in the fibrocytes associated with generation of the ion gradients between the endolymph and perilymph.

Rapid and transient up-regulation of Na+/myo-inositol cotransporter transcription in the brain of acute hypernatremic rats.

The osmoregulatory system is well developed in the brain. Osmolytes contribute to maintenance of cell volume and cellular functions without changing intracellular ionic composition. Myo-inositol is regarded as one of the major osmolytes in the brain. In the present study, we investigated the changes in expressions of sodium myo-inositol cotransporter (SMIT) mRNA in the brain of acute hypernatremic rats by in-situ hybridization and Northern blot methods. Under moderate acute hypernatremic conditions, SMIT mRNA level increased markedly at 1 h and returned to almost control levels at 3 h, in accordance with plasma Na+ concentrations. Especially, distinct increases in SMIT mRNA expression were observed in the granule cells and glial cells in the cerebellum. These findings indicate that SMIT plays an important role in osmoregulation, especially in the early stages of acute hypernatremia in the brain.

Expression of Na+/myo-inositol cotransporter mRNA in normal and hypertonic stress rat eyes.

We studied the localization of Na+/myo-inositol cotransporter (SMIT) mRNA in normal and hypertonic stress rat eyes by in situ hybridization histochemistry using cRNA probes. SMIT mRNA signals were observed in the iris-ciliary body, the lens epithelial cells, and the ganglion cell layer and the inner nuclear layer of the retina. There was a rapid increase on SMIT mRNA in the retina of hypertonic stress rats compared with control rats. These findings suggest that Na+/myo-inositol cotransporter gene expression is osmotically regulated in vivo to protect retinal neuronal function against hypertonic stress.

The differential osmoregulation and localization of taurine transporter mRNA and Na+/myo-inositol cotransporter mRNA in rat eyes.

We studied the cellular localization and osmotic regulation of taurine transporter (TauT) mRNA in the rat eyes using in situ hybridization. TauT mRNA signals were expressed in the ciliary body, and the outer part of the inner nuclear layer (INL), the outer nuclear layer (ONL) and the inner segment (IS) of the adult rat retina. Chronic hypernatrema, induced by gavaging with 1 ml/100 g body weight of 5% NaCl every other day for 7 days, markedly increased in TauT mRNA in the retina compared with control rats. However, there was little change in TauT mRNA in the eyes in acute hypernatremic state that is induced by single injection of high concentration of NaCl. On the contrary, acute hypernatremic rats displayed markedly elevated Na+/myo-inositol cotransporter (SMIT) mRNA in the retina and the iris-ciliary body and the lens epithelium. Under chronic hypernatremic conditions, there was no significant increase in SMIT mRNA in rat eyes. These findings suggest that TauT mRNA is osmotically regulated in vivo to protect retinal neuronal function, especially against chronic hypernatremic conditions, in contrast to rapid up-regulation of SMIT mRNA in acute hypernatremic rats.

Expression of c-fos and c-jun mRNA following transient retinal ischemia: an approach using ligation of the retinal central artery in the rat.

The expression of the proto-oncogenes c-fos and c-jun was examined by in situ hybridization at various timepoints following transient retinal ischemia by means of ligation of the retinal central artery in the rat. Ischemia of 90-minute duration resulted in the degeneration of neurons in both the ganglion cell layer and the inner nuclear layer at 48 hours after reperfusion. The expression of c-fos and c-jun messenger RNA throughout the entire inner nuclear layer was transiently coinduced following 90-minute retinal ischemia with a peak at 1 hour after reperfusion. This expression was also found in the ganglion cell layer at 3 hours after reperfusion. Weak signals for c-fos and c-jun mRNA were observed at 24 hours after reperfusion and returned to near control levels by 48 hours. c-jun protein expression was detected in the ganglion cell layer, the middle of the inner nuclear layer, and optic nerve head at 3 hours, but not 1 hour, after lethal ischemia/reperfusion; however, c-fos protein expression was not detected after reperfusion. Whereas no neuronal degenerative changes were found at 7 days after 30-minute ischemic retina, c-fos and c-jun messenger RNA were also induced at 1 hour postreperfusion. To our knowledge, this study is the first report to show expression patterns of immediate-early genes after retinal ischemia/reperfusion. These results suggest that changes in expression of c-fos and c-jun after transient retinal ischemia are similar to those after transient brain ischemia, and the selective occlusion of the central retinal artery will provide a useful model for studying ischemic neuronal degeneration in vivo in the rat retina.

Cellular localization of Na+/MYO-inositol co-transporter mRNA in the rat brain.

The distribution of Na+/MYO-inositol co-transporter (SMIT) mRNA in the rat brain was studied by in situ hybridization histochemistry. The highest levels of SMIT mRNA were observed in the choroid plexus. Intense hybridization signals were found in the pineal gland, the area postrema, the hippocampus, the locus coeruleus, the suprachiasmatic nucleus, the olfactory bulb and the Purkinje cell and granule cell layers of the cerebellum. Low to moderate levels of labelling were detected in almost all neurones and small glia-like cells throughout the brain. These results suggest that almost all cells in the brain possess an SMIT-mediated osmotic and ionic regulatory system, and uneven densities of positive SMIT mRNA signals may reflect the differences in sensitivity of the cells to osmotic and ionic changes and also reflect differences in permeability of capillaries.

GLAST mRNA expression in the periventricular area of experimental hydrocephalus.

Glutamate transporters play an important role in maintaining the extracellular glutamate concentration below the neurotoxic level. We investigated the expression of glutamate/aspartate transporter (GLAST) mRNA in the periventricular region of rats with kaolin-induced hydrocephalus by in situ hybridization (ISH). The density of GLAST mRNA-positive cells and the level of hybridization signals per positive cell significantly increased in the acute stage of hydrocephalus. We also demonstrated co-localization of GLAST mRNA and GFAP immunoreactivity in a single cell using the combined methods of ISH and immunohistochemistry. These findings suggest that GLAST is expressed in the reactive astrocytes of the periventricular area and regulates extracellular glutamate concentration after hydrocephalic brain injury.

The spectrum of beta ig-h3 gene mutations in Japanese patients with corneal dystrophy.

PURPOSE: This study was undertaken to identify beta ig-h3 gene mutations in Japanese patients with granular corneal dystrophy (GCD), Avellino corneal dystrophy (ACD), lattice corneal dystrophy (LCD), and Reis-Bücklers' corneal dystrophy (RBCD). R124H, R124C, R555W, and R555Q mutations have been reported in Europe to cause ACD, LCD type I, GCD, and RBCD, respectively. METHODS: In total, 91 Japanese patients who had been clinically diagnosed with GCD, LCD, or RBCD were investigated to determine whether they had mutations in the beta ig-h3 gene. Genomic DNA was amplified using the polymerase chain reaction and analyzed using single-strand conformation polymorphism techniques. Mutations were identified using the direct sequencing method. RESULTS: In 68 unrelated patients who had been diagnosed with GCD, 62 patients (91%) were found to have the R124H mutation, which has been reported to cause ACD, whereas only six patients (9%) had the R555W mutation. In LCD patients, 10 patients with type I disease had the R124C mutation, and 10 patients with type IIIA disease had a P501T mutation. One patient with atypical LCD had an L527R mutation. In two patients with RBCD, one had an R555Q mutation and the other patient with geographic opacities was found to have an R124L mutation. CONCLUSIONS: Depending on the specific mutation in the beta ig-h3 gene, the phenotypes of corneal dystrophy may differ. Our results indicate that assay of mutations in the beta ig-h3 gene is required to establish a correct diagnosis.

Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or leber congenital amaurosis.

RPE65 is a protein of unknown function expressed specifically by the retinal pigment epithelium. We examined all 14 exons of this gene in 147 unrelated patients with autosomal recessive retinitis pigmentosa (RP), in 15 patients with isolate RP, and in 45 patients with Leber congenital amaurosis (LCA). Sequence anomalies that were likely to be pathogenic were found in two patients with recessive RP, in one patient with isolate RP recategorized as recessive, and in seven patients with LCA. Cosegregation analysis in each available family showed that all affected individuals were either homozygotes or compound heterozygotes and that all unaffected individuals were either heterozygote carriers or homozygous wild type. In one family, there was one instance of a new mutation not present in either parent of the affected individual. In another family, affected members with recessive RP in three branches (i.e., three distinct pairs of parents) were compound heterozygotes for the same two mutations or homozygous for one of them. Based on our results, mutations in the RPE65 gene appear to account for approximately 2% of cases of recessive RP and approximately 16% of cases of LCA.