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Because their spectral sensitivity shows that ultraviolet [UV] light reaches the retina, 1 we utilized juvenile goldfish [Carassius auratus] to study light damage induced by 300-400nm light at about 30μWcm-2. Electron microscopy [EM] and the electroretinogram [ERG] were used to assay retinal health. Cone ellipsoids in the vicinity of mitochondria were tortuous and swollen at 3 days of such near-UV treatment. Double cones showed some outer segment damage. Curiously, at 6 days, some mitochondria were displaced toward the external limiting membrane in double cones. By 12 days, the double cone outer segments were nearly gone, and the mitochondrial displacement extended to single cones [including UV cones]. Inner segments were filled with a smooth reticulum. Rods were normal. Although there was ultimately [by 32 days] a preferential loss of UV visual sensitivity, at 12 days a UV sensitivity peak remained which could be isolated by chromatic adaptation, suggesting that UV cones continued to function. Widespread cone damage by UV light suggests a mechanism independent of visual pigment absorbance, though the delayed loss of UV sensitivity in UV cones may be mediated through their UV rhodopsin. Retinoic acid had been implicated in protection of photoreceptors from light damage, retinal development, and gene transcription. All-trans retinoic acid [1 μM] was added to the aquarium. Treatment of 3-day-UV fish with retinoic acid for 5hr and of 12-day-UV fish for 7hr reversed many of the signs of UV damage. [ABSTRACT FROM AUTHOR]

Background: Although the mutations of the REP-1 gene were reported in the Caucasian patients with choroideremia, no report was found in non-Caucasian patient except our previous case report. We performed multi-institutional study of the REP-1 gene in Japanese patients with choroideremia. Methods: Twenty four unrelated Japanese families with choroideremia were analyzed. It included 24 patients and a carrier. The method for screening was a nonradioisotopic modification of single strand conformation polymorphism (SSCP). The PCR products from the patients and the carrier were screened and directly sequenced using an automated DNA sequencer. Results: SSCP analysis showed abnormal band shifts in the patients and the carrier. Direct sequence analysis showed 15 kinds of mutations in 19 families. They included 8 nonsense mutations, 7 frameshift mutations, and 4 point mutations in the splice site. Conclusion: The mutations in a racial background different from families reported before confirm that the REP-1 gene defect causes choroideremia. Most of Japanese patients with choroideremia are caused by the REP-1 gene mutations. No missense mutation has been found in Japanese patients either. Although large deletion was sometimes recognized in European patients, we could not find it in this series. [ABSTRACT FROM AUTHOR]

RPE65 encodes a retinal pigment epithelium (RPE)-specific protein thought to be involved in the 11 -cis retinoid metabolism, a key process in vision.To evaluate the visual implications of defects in this gene, we screened patients with various types of inherited retinal dystrophies. We found two nonsense mutations, 1121delA and R234X, in two sibs with Leber's congenital amaurosis. The presence of either one of the two mutations on each allele indicates that they were compound heterozygotes. In a subsequent screening, two missense mutations, L22P and H68Y, were found in a patient with autosomal recessive retinitis pigmentosa.This individual was also a compound heterozygote. These results suggest that the level of impairment of the RPE65 protein, presumably abnormal activity in the case of amino acid substitutions and absence of protein in the case of null mutations, controls the rate at which the photoreceptors degenerate, and hence the severity of the phenotype. [ABSTRACT FROM AUTHOR]

The turnover of phototransductive membrane is an essential process for the viability of photoreceptor cells. Molecular motors are likely candidates for the trafficking of phototransductive membrane during various stages of its turnover. Our studies of molecular motors in photoreceptor cells and the RPE are aimed at understanding mechanisms involved in phototransductive membrane turnover and the cellular basis of some forms of retinal degeneration. Results described here represent initial studies on the role of myosin VIIa in the retina. Defects in the myosin VIIa, gene have been found to cause forms of Usher syndrome. Myosin VIIa, was localized in the apical processes of the RPE and in the connecting cilium of the photoreceptor cells. In the retinas of shaker-1 mice, which express mutant myosin VIIa, the only identifiable structural defect was the mislocalization of the melanosomes of the RPE. Disk membrane shedding did not appear to be affected. Nevertheless, the localization of myosin VIIa, in the photoreceptor cilium suggests that the protein might be involved in transporting phototransductive membrane to the outer segment. [ABSTRACT FROM AUTHOR]

Defects in the gene encoding for the unconventional myosin VIIa leads to human Usher syndrome 1B, the most common form of hereditary combined blindness and deafness. To determine cellular function of myosin VIIa, we have investigated the subcellular localization of myosin VIIa in spacial relation relationship to potentially interacting proteins in mammalian photoreceptor cells. Western blot analysis of the axonemal fraction of photoreceptor cells by Western blot show that myosin VIIa and actin, as well as opsin, were present in the ciliary portion of the photoreceptors. Improved immunoelectron microscopy revealed that in mammalian photoreceptor cells, myosin VIIa was localized at the membrane of the connecting cilium linking photoreceptor inner segments with their outer segments. In addition, actin, the functional partner of myosins, actin, was detected at the ciliary membrane providing considerable evidence that myosin VIIa also interacts with actin filaments in the connecting cilium. Applying the highly-sensitive silver-enhanced indirect nanogold™-labeling for immunoelectron microscopy, intense anti-opsin immunoreactivity was recognized in the membrane of the connecting cilium indicating that rhodopsin trafficing to the outer segment occurs through the ciliary membrane. Co-localization of rhodopsin with the myosin VIIa-actin systems at the membrane also provides evidence for mysion VIIa driven ciliary transport of rhodopsin. Absence of myosin VIIa or presence of non-functioning myosin VIIa may lead to defects in the ciliary transport and cause retinitis pigmentosa in Usher syndrome 1B patients. [ABSTRACT FROM AUTHOR]

Cryopreserved retinal pigment epithelial (RPE) cells at -80°C can be used for transplantation studies. Long-term viability at -196°C has been used for many other cell lines. The origin and purity of human RPE cell lines were assessed by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR) using RPE cell markers [cytokeratin, cellular retinaldehyde binding protein (CRALBP), tyrosinase, tyrosinase-related proteins I and II (TRP-I and II), Na,K-ATPase α1 and β2]. Cultured human RPE cell lines were cryopreserved at -80°C and -196°C. After cryopreservation, human RPE cells were re-cultured on laminin-coated polystyrene dishes and laminin-coated collagen sheets. Differentiation was evaluated by the expression level of RPE marker genes using RT-PCR. It was found that cryopreserved human RPE cells on laminin-coated collagen sheets disclosed relatively strong re-expression of their marker genes if compared to human RPE cells cultured on laminin-coated polystyrene dishes. Cryopreservation of RPE cells at both temperatures, thawing and subsequent culturing on laminin-coated collagen sheets can enhance the feasibility of using cryopreserved RPE cells for transplantation. [ABSTRACT FROM AUTHOR]

To establish auto iris pigment epithelial (IPE) cell transplantation, we examined monkey IPE and performed auto IPE transplantation. IPE cells of the monkeys were obtained by peripheral iridectomy and cultured as we previously reported. Immunocytochemical study was also performed to confirm that they were epithelial in origin. Cultured condition were also studied with bovine, rabbit, mouse, auto, or human serum. Transplantation study was performed by transvitreal approach in the monkey eyes. Mouse and rabbit serums were extremely toxic to the monkey IPE cell culture. Conversely, the cells grew well in the medium with bovine, monkey, and human serum. The difference was not statistically significant among bovine, monkey, and human serum. We demonstrated the presence of the transplanted cultured IPE cells after 60 days of transplantation by ophthalmoscopical and histochemical examination. Several reports also have demonstrated that IPE cells may have about 80% phagocytic function and may also be able to form tight junctions in the subretinal space. The establishment of auto IPE cell transplantation may improve the problem of the rejection. [ABSTRACT FROM AUTHOR]

Melatonin is an important retinal neurohormone involved in rhythmic photoreceptor metabolism and in regulating the release of retinal dopamine. Systemic administration of melatonin and conditions supporting its synthesis in the retina seem to be related to increased light damage susceptibility. We found that intraocular injection of competitive melatonin receptor antagonists prior to the dark period before bright light exposure resulted in substantial protection of photoreceptors from light damage. Photoreceptor cell structure was much better preserved in antagonist injected eyes and the ERG threshold was approximately 2 log units of intensity lower. Intraocular injection of a number of other compounds which are known to affect endogenous dopamine levels or stimulate or block dopamine receptors did not have a significant effect on the ERG. However, some showed a tendency to reduce or enhance the effect of light damage on the ERG depending on their expected effect on dopamine levels. These results indicate that blocking the action of endogenous melatonin at retinal receptors can protect photoreceptors from damage by light. Further work is needed to investigate a possible role for dopamine in this protection. [ABSTRACT FROM AUTHOR]

We have been interested in cellular regulatory mechanisms operating in mammalian retinal neurons, especially those determining sensitivity to factors which either promote or inhibit cell death. The production of reactive oxygen species through oxidative stress is believed to be an important mediator of neuronal cell death although the precise mechanism by which this occurs is unknown. Pigment epithelia derived factor (PEDF) has been shown to promotes neurotrophic differentiation and the survival of neurons of the central nervous system. Using a cell culture system, we demonstrate that multiple populations of retinal neurons can differentiate and be maintained in a chemically defined environment and that cell death induced by reactive oxygen species can be quantitated. The data also show that this induced death is dose dependent and occurs by an apoptotic mechanism which can be inhibited by PEDF. The mechanism by which PEDF provides this protection may be very important for inhibiting the apoptotic death of retinal neurons which occurs in retinitis pigmentosa, macular degeneration, glaucoma or other neurodegenerative diseases. These cultured neurons will also be useful for analyzing apoptotic responses of each of the retinal neuron cell types to a variety of "cytotoxic" agents and to the rescue effect of a number of "factors" either alone or in combination. [ABSTRACT FROM AUTHOR]

Pigment epithelium-derived factor (PEDF) was first described as a secreted product of human fetal retinal pigment epithelial (RPE) cells in culture with a potent neuronal differentiating activity on human retinoblastoma Y-79 cells.1 It was reported that PEDF at nanomolar concentrations induces a morphological differentiation of Y-79 cells, characterized by the elongation of neurite-like processes and the expression of neuronal markers, e.g., neuron-specific enolase and the 200-kDa neurofilament subunit.2 It was shown later that PEDF can also induce the differentiation of cells from another human retinoblastoma, the Weri cells.3 While its role in vivo on retina cells is not known yet, its origin and these activities suggest that PEDF may play a role as a neurotrophic factor for the retina. In this regard, evidence of its effects on neurons from the CNS and PNS is accumulating rapidly and supports the notion that PEDF is a multipotent neurotrophic factor that acts upon various types of neurons and from different species. For example, it promotes the survival of rat cerebellar granule cell (CGC) neurons in culture,4 including protection against glutamate neurotoxicity5 and against both natural and induced apoptosis in vitro;6 it promotes survival and differentiation of developing avian and murine spinal motor neurons.7 Given the important biological activities of PEDF, it is of interest to investigate its mechanism of action on the retina. Therefore, the overall objective of this review will be to discuss the biochemistry of PEDF in the context of a potential use as a relevant neurotrophic factor for the retina. [ABSTRACT FROM AUTHOR]

To investigate possible trophic effects of transplanted rod photoreceptors upon recipient cone photoreceptors in mutant retinal degeneration (rd) mice retinas. [ABSTRACT FROM AUTHOR]

The feasibility of a retinal prosthesis for the blind has been discussed since the 1950s. Early attempts to realize a subretinal implantable prosthesis suffered from technical and microsurgical limitations at that time. Nowadays these problems can be solved because of the amazing progress that has been made in the fields of intraocular surgery, silicon microfabrication and our knowledge of the silicon/neuron junction. Therefore, a new attempt to develop a retinal prosthesis seems justified. While other research groups1-3 have concentrated their efforts on the development of an epiretinal visual prosthesis, which receives energy and preprocessed visual information via a relatively complex telemetric system, Chow and coworkers4-7 published the concept of a retinal implant consisting of thousands of microphotodiodes. The principle of this concept is based on an implant that is placed in subretinal space and stimulates the underlying retinal cells with the photocurrents generated solely from the incident light (Fig. 1). [ABSTRACT FROM AUTHOR]

For blind subjects with retina degenerative diseases (especially: retinitis pigmentosa and macular degeneration) to regain visual perception, a team of 14 expert groups develops a partially implantable, learning visual prosthesis (retina implant). This team with experts from several biological, medical, and technological areas is supported by the German research ministry (BMBF) and is coordinated by the author. Retina implants consist of a learning retina encoder (RE)—to be mounted on a frame of glasses or embedded in a contact lens—for the approximate simulation of parts of the retina by transforming light patterns into impulse trains similar to the receptive field properties of ganglion cells, a microcontact foil as retina stimulator (RS) to be implanted adjacent to the ganglion cell layer, and a wireless signal- and energy transmission between RE and RS. The function of the various spatiotemporal filters of the RE, which is being implemented by learning neural nets, will be tuned individually in a dialog with the implant-carrying subject for optimal visual perception. The development and successful test of retina implant prototypes in animals is expected at the end of the first 4-years research phase in 1999. In a subsequent research phase with participation from industry, the next step for adaptation of the retina implant system for application in humans and first trial tests with a small number of volunteers will follow. It is expected that implant-carrying subjects will be able to recognize position and ‘gestalt' of larger objects (e.g. window, door, chair, table) based on RE and RS with about 500 microcontacts in connexion with retinal ganglion cells, and that they will be able to walk and orient themselves without help in most unknown environments. This hope is partly based on recent findings that simple gestalt perceptions could already be elicited in several blind subjects by temporary microstimulation of retinal ganglion cells. Furthermore, anatomical studies have recently shown that a significant portion of the ganglion cells and the optic nerve in this group of blind subjects remain intact, even though the layer of photoreceptors is degenerated. [ABSTRACT FROM AUTHOR]

The visual prosthesis being developed will provide useful vision to people blind because of photoreceptor loss due to retinal degenerative diseases such as age related macular degeneration (AMD) and retinitis pigmentosa (RP). A retinal prosthesis will replace photoreceptor function with an electronic device. In a healthy retina the pho- toreceptors initiate a neural signal in response to light. Photoreceptors are almost com- pletely absent in the retina of end-stage RP patients and in the macular region of AMD patients. However, cells to which photoreceptors normally synapse, (i.e. bipolar cells) survive at high rates. In blind volunteers, we have shown that controlled electrical signals applied with a microelectrode positioned near the retina elicit the perception of a spot of light that correlates both spatially and temporally to the applied stimulus.1,2 When multiple electrodes are activated in a two dimensional electrode array, a number of small spots of light are perceived by the patient(s) which when viewed together form an image representative of the pattern of active electrodes. Thus similar to how an image is formed by a dot-matrix printer, when controlled pattern electrical stimulation of the remaining retinal neurons is coupled with an extraocular image acquisition and transmission system, it could allow blind patients to regain form vision. [ABSTRACT FROM AUTHOR]

Presently, there is no treatment by which to restore vision to patients blinded by outer retinal diseases such as retinitis pigmentosa and age-related macular degeneration. We have developed a retinal prosthetic that is designed to be placed in the sub-retinal space and thereby stimulate second-order retinal neurons spared by disorders that induce selective photoreceptor degeneration. The device is semiconductor-based and does not require an external power supply for operation. Previous studies have characterized the device with respect to biocompatibility and function. The present study concerns cortical potentials elicited when the implant is stimulated with infrared light, to which the native retina is insensitive. The results indicate that these potentials share several response characteristics with conventional visual evoked potentials and support the conclusion that the implant is capable of electrically activating the visual system. [ABSTRACT FROM AUTHOR]

Diseases of the retina and optic nerve are common causes of irreversible blind- ness. Given the lack of effective treatments, several laboratories are utilizing micro- electronic technology to develop either a cortical or retinal prosthesis. Each strategy offers certain advantages, but both face numerous and formidable challenges. Conse- quently, a clinically useful device of either type is still conceptual. The technological means to build prostheses are available but the ultimate obstacle is the integration of the technology with the brain. This review primarily focuses on our efforts to develop a retinal prosthesis. In particular we address the two problems that we believe to be most challenging: 1) need to demonstrate that retinal stimulation can produce "useful" vision in a blind patient, and 2) need to demonstrate long-term biocomopatibility of an implanted device in an animal. Brindley produced crude visual sensations by stimulating the visual cortex in a blind patient 32 years ago.1 This early success coupled with the broad therapeutic poten- tial of a cortical prosthesis to treat blindness caused by damage of either the retina or optic nerve has made this pursuit attractive to many scientists. Developing a cortical prosthesis that remains functional and biocompatible for prolonged periods of time is a difficult task and progress has not surprisingly been slow.2 The convoluted surface of the mobile brain and the need for penetration of the subsurface layer of the visual cortex increase the difficulty of maintaining a stable interface. The potential risks of neurosurgery are also a formidable if not simply a psychological barrier for patients. [ABSTRACT FROM AUTHOR]

The spatial distribution of cyclic nucleotide gated (CNG) channel molecules in photoreceptor outer segments (OS) dissociated from amphibian retinas was investigated by performing immunofluorescent localization of spectrin and using the known spectrin immunoreactivity of the beta subunit of the channel in rods to infer the location of CNG channels. In the OS of rods and cones, anti-spectrin immunoreactivity occurred as a bright streak of fluorescence at the ciliary axoneme. Rod OS displayed an additional pattern of staining not present in cone OS, namely a series of thin, discrete, longitudinal lines of fluorescence that extended the entire length of the OS and coincided with incisures. Thus, the location of immunoreactivity to spectrin in the OS of both photoreceptor cell types coincided with locations known to contain arrays of longitudinally-oriented microtubules. These findings provide strong evidence that CNG channel molecules are confined within OS membranes to specific restricted locations in the immediate vicinity of microtubules, eg., CNG channel molecules may be tethered to microtubules via the spectrin-like portion of their beta subunits. Because the localization of CNG channels within photoreceptor OS is expected to influence the spatiotemporal dynamics of phototransduction and adaptation, the diverse localization of channels within the OS could contribute to the different functional properties of rods and cones. Because evidence suggests that the OS of human and amphibian photoreceptors have similar microtubule-containing cytoskeletal systems at similar locations, the spatial distribution of CNG channel molecules described here for amphibian photoreceptor OS is also expected to occur in human photoreceptor OS. A disturbance in the localization of CNG channels, or in their associations with other molecules or microtubules, within photoreceptor OS is expected to disturb OS structure and function, which may be relevant for some human retinal degenerations. [ABSTRACT FROM AUTHOR]

The distribution of hyaluronan (HA) in the interphotoreceptor matrix (IPM) was evaluated in human tissues using a specific probe for HA (bHABC, biotinylated hyaluronan binding complex). In tissue sections, bHABC heavily decorates HA in the matrix surrounding rods and shows lighter binding to the cone associated matrix. Both HA and the glycoprotein SPACR are present in insoluble IPM isolates removed from unfixed retinas with distilled water treatment. To determine whether SPACR associates with HA, precipitation experiments were performed using the detergent cetylpyridinium chloride (CPC), in conjunction with hyaluronidase digestions. SPACR is present in the CPC pellet when an undigested IPM sample is precipitated, but is not present in the CPC pellet when the IPM is first digested with Streptomyces hyaluronidase. These results indicate that SPACR binds to HA, and implicates HA as the primary scaffold onto which IPM molecules form associative interactions in the IPM. [ABSTRACT FROM AUTHOR]

Phosducin (Phd) is a soluble phosphoprotein, selectively binding to the beta-gamma (βγ) complex of G-proteins, playing a role in intracellular signaling processes. We have molecularly identified 3 Phd isoforms expressed in the human retina. Based on their ability or inability to interact in our assay system with Gβγ proteins, we named them Phosducin-Like Protein 1 (PhLP1) or Phosducin-Like Orphan Proteins (PhLOP1 & PhLOP2), respectively. To further study these Phd isoforms and their potential involvement in normal and abnormal visual processes through genetic alterations, we identified and characterized an 85 kb P1-PhLP human genomic clone that contains PhLPl and PhLOP1. With fluorescence in situ hybridization (FISH) to human metaphase chromosomes, this P1-PhLP genomic clone was mapped to human chromosome 1q25.3. Previous work identified a gene locus for phosducin (PDC) to 1q25-q31.1 by somatic cell hybridization and in situ hybridization. Within the P1-PhLP genomic clone, the previously characterized complete PDC gene was identified; furthermore, the 5'-flanking region and a potential promoter region of PhLOP1 was identified between Exon 2 and Exon 3 of the PDC gene. Initial transfection experiments with luciferase activity reporter PhLOP1 constructs, ranging in size from 386 to 852 basepairs (bp), suggest that no tissue specific retinal promoter activity is contained in this 5'-flanking region of the PhLOP1 when compared to the high retinal specific promoter activity observed for a 123bp construct for Interphotoreceptor Retinoid-Binding Protein (IRBP). Alternatively, a represser element is still present and preventing promoter activity in the PhLOP1 constructs. These results correspond to the low levels of mRNA of the PhLOP1 observed in the retina compared to Phd. These data suggest that both PhLP1 and PhLOP1 are created through alternative splicing of the Phd gene and that a single PDC gene at 1q25.3 is responsible for these three retinal isoforms. [ABSTRACT FROM AUTHOR]

To investigate the role of rds/J.peripherin in photoreceptor outer segment membrane assembly, we have transfected embryonic Xenopus laevis retinas with phosphorothioated antisense or sense RNA complementary to specific regions of the three Xenopus rds/peripherin homologs. We have demonstrated that using antisense oligonucleotides complementary to the three homologs of rds/peripherin, we can significantly alter the structure of photoreceptor outer and inner segments. In addition, the immunolocalization patterns using xrds35 and -38 anti-peptide sera and an anti-opsin monoclonal antibody were significantly reduced, suggestive of aberrant outer segment membrane assembly. We have successfully generated a model upon which to study the functional role of rds/peripherin, the product of the rds gene, by down-regulating the expression of this protein in developing photoreceptors. A better understanding of rds/peripherin gene expression will shed light on the role of this molecule in rod and cone photoreceptor outer segment development, structure, function and survival. This will also provide additional insights into the mechanisms of, and possibly open new therapeutic avenues for, the retinal degenerative processes taking place in the heterogeneous clinical patterns so far described in association with peripherinJ.RDS human gene mutations in association with autosomal dominant retinitis pigmentosa. [ABSTRACT FROM AUTHOR]

It is well known that the retina can be stimulated in vitro and in vivo with monopolar transretinal or local bipolar applied electric current.1-4 These results indi- cate that it might be possible to develop a retina implant that electrically stimulates the remaining cells of the retinal network electrically, even if the photoreceptors are completely degenerated. Current research focuses on two different approaches for the development of an intraocular prosthesis for patients suffering from photoreceptor degeneration. Several groups favour direct electrical stimulation of retinal ganglion cell5-7 from the epiretinal side while Tassiker and Chow proposed in patents to stim- ulate neurons of the inner nuclear layer8-10 from the subretinal side (see also Zrenner et al. in this volume). [ABSTRACT FROM AUTHOR]

Retinitis pigmentosa (RP) is one of the most frequent hereditary dystrophies. Multiple genetic defects have been found to be expressed in the photoreceptors and retinal pigment epithelium (RPE) cells of RP patients. The Royal College of Surgeons (RCS) rat is the animal model most commonly used to study RP. In the RCS rat, the photoreceptors begin to degenerate during the third postnatal week, and retinal degeneration continues toward the inner retina after their loss. We used a subtractive hybridization technique to identify differentially expressed genes that might be responsible for the induction of degenerative processes in the RCS retina. Until now, we have isolated about 30 clones with putative differentially expressed sequences. To date, two of these sequences were confirmed to be differentially expressed in the RCS rat but not in control rats. The method described here is therefore suitable for identifying differentially regulated genes, not only in RP retinas, but also in other retinal dystrophies and may help to further elucidate the pathophysiology of retinal diseases. [ABSTRACT FROM AUTHOR]

This project aimed to investigate whether the overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium (RPE), induced by recombinant adenovirus mediated delivery of VEGF164, is sufficient to initiate the development of leaky choroidal blood vessels in a rat model. Eyes of pigmented rats were subjected to krypton laser photocoagulation or subretinal injection with recombinant adenovirus constructs Ad.RSV.VEGF or Ad.CMV.VEGF. Initially the animals were examined with fundus photography followed by fluorescein angiography. Retinal photographs demonstrated that the krypton laser produced moderate burns and that the subretinal injection resulted in the formation of a bleb in the subretinal space. Injection of control recombinant adenovirus Ad. RSV.βgal resulted in the expression of βgal reporter gene in the RPE layer. Of the 141 laser spots delivered into a total of 17 rat eyes, 52.4% developed fluorescein leakage that was detectable by angiography. Of seven eyes injected with Ad.RSV.VEGF, 5 developed leakage at 2 weeks post injection, and of 8 eyes which were injected with Ad.CMV.VEGF, 5 developed leakage at 1 week post injection. However, the development of leaky blood vessels was temporary and both Ad.RSV.VEGF and Ad.CMV.VEGF injected animals showed regression of leaky vessels by 4 and 5 weeks post injection, respectively. These results suggest that the upregulation of VEGF in the RPE layer may be a key factor in the series of events leading to the development of CNV. [ABSTRACT FROM AUTHOR]

N-methyl-N-nitrosourea (MNU) showed a toxic effect on the mouse and rat retinal photoreceptor cells leading to apoptosis. In agreement to human retinitis pigmentosa, the primary event was photoreceptor apoptosis. After photoreceptor cell loss, retinal pigment epithelial (RPE) cell migration to inner retina and accumulation around blood vessels were seen in humans; in rats, the migration was seen, but they were not accumulated around blood vessels, and in mice, RPE migration was not seen. In C3H mice carrying the rd gene as well as C3H mutants (Fas/Fas ligand-deficient mice), retinal degeneration occurred by an apoptotic mechanism. Therefore, Fas/Fas ligand do not play a primary role in the photoreceptor apoptosis in rd gene carrying mice. [ABSTRACT FROM AUTHOR]

Glutathione is a potent endogenous scavenger of oxygen free radicals in living cells. Decreased levels of glutathione are involved in aging and some neurodegenerative diseases. To investigate an effect of glutathione depletion in the horizontal cells of carp retina, the gap junctional intercellular communication between horizontal cells was studied using both techniques of intracellular recording of light-induced responses and dye coupling of the fluorescence, Lucifer Yellow (LY). The glutathione depletion was accomplished by single injection of 2.5μmol L-buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. Intravitreal injection of BSO induced a dramatic reduction of retinal level of glutathione 2 days after treatment. The low level of glutathione continued for a further 4-5 days and thereafter was gradually recovered to about 70% of the control level by 50 days after injection. In normal retinas, the response amplitude of horizontal cells was monotonically enhanced as the diameter of the spot increased (0.5-4.0mm) and correspondingly the dye diffusion area was wide, as the injected LY normally diffused to several neighboring cells. Depletion of glutathione by BSO significantly altered the spatial properties of horizontal cells by increasing the response amplitude to central spots and slightly decreasing that to peripheral annuli, which were observed by 4 days after treatment. It also restricted intracellular LY to one or two cells. Accompanying the recovery of the cellular level of glutathione, the spatial properties and dye coupling of horizontal cells were recovered to normal. These alterations of spatial and dye coupling properties of horizontal cells were well correlated with accumulation of reactive oxygen species (H2O2) during glutathione depletion. The present data have demonstrated for the first time that neuronal gap junctional channels can be modified by intracellular glutathione via oxidative stress. [ABSTRACT FROM AUTHOR]

Earlier work from our laboratory showed that retinal rods of albino rats closely regulate the number of photons they absorb. This "photostasis number" is maintained at about 1016 per eye per day, regardless of the light intensity in the habitat or day length. We showed that the retina is plastic and that the regulation is achieved by modification of the optical density of rod cells. In the present study, we showed that the eyes of pigmented rats also maintain photostasis. Total rhodopsin in the dark-adapted eye is higher in pigmented than in albino rats. Replicating the lower levels seen in albino rats requires either pupillary dilation or use of light intensity that approaches that of outdoor lighting. Under those conditions, rod cell count is also significantly lower than that at light intensities normally used in animal colonies. Despite these variations in retinal light-absorbing properties, all animals absorbed the photostasis number of photons (1.4 × 1016 per eye per day), 13,000-15,000 photons per average rod per sec. This range of rates is well within that known to saturate mammalian rods and keep them unresponsive to further absorption. We therefore hypothesize that a function of photostasis is to render rods functionless during daylight hours, but this hypothesis requires in addition that most if not all rods absorb at these rates. To test this possibility, we measured the rate of photon absorptions across a wide range of retinal areas with an ocular transmission photometer (OTP) and found that most, if not all, rods are bsorbing at these rates. An interesting aspect of the OTP studies is that excised, pigmented rat eyes retain pupillary responses. These responses are circadian in nature: they anticipate light-period boundaries and shift only gradually after shifts in the photoperiod. [ABSTRACT FROM AUTHOR]

Albino rainbow trout, Oncorhynchus mykiss, are resistant to light damage, losing only their rod outer segments (ROS) in full daylight (10,000 to 100,000 lux) at 11°C. 1 To compare light damage in albino trout with light damage in albino rodents, we analyzed central retinal structure in albino trout exposed to full daylight, indoor dim day-light (30 lux-30 days or longer) and strong constant light (3000 lux). In albinos remaining outdoors or placed in constant light, ROS volume was reduced but the number of photoreceptor nuclei did not decline. Albinos placed in dim daylight re-grew ROS to 60% or more of normal volume but when returned to outdoor raceways lost most of their ROS volume within 5 days. Outdoor albinos placed in dim daylight replaced ROS much more slowly. In neither case was there a change in number of photoreceptor nuclei. In affected albinos there is apparently little rod cell death during the initial phases of light insult to ROS or thereafter. This confirms that most rod cells with ROS damage survive and retain capacity to re-grow ROS, and any which undergo apoptosis are replaced by cells derived from rod progenitors. To ascertain whether the 11°C temperatures at which trout were held was protective, we exposed albino and normal oscars, Astronotus oscellatus, to 3000 lux constant light at 28°C. Albinos lost -50% of their rod nuclei over 14 days and normals appeared unaffected. Thus, retinal photo-degeneration in 3000lux constant light in albino trout at 11°C was limited to ROS loss, whereas ROS destruction lead to loss of rod cell bodies in albino oscars at 28°C. The ability to use ambient temperature to preclude or permit light damage to proceed to cell death in a cone rich, diurnal retina could be exploited to study mechanisms for susceptibility to and resistance to photo-degeneration. [ABSTRACT FROM AUTHOR]

Age and monocular enucleation have been examined as potential determinants of the susceptibility of the retina to light damage. BALB/c and C57BL/6J -c2J mice were examined. BALB/c mice showed an initial increase in susceptibility to light damage between 8.5-10 weeks of age. This did not coincide with the time of sexual maturation, as reported previously for the rat. C57BL/6J-C2J mice, which are significantly more resistant to light damage than most other albino strains, showed no age-related change in susceptibility. Monocular enucleation had no effect on the susceptibility of the intact eye to light damage in either strain. [ABSTRACT FROM AUTHOR]

The main theme of this volume is the inherited retinal degenerations, but the most important causes of blindness have a different aetiology, not directly related to genetic defects. Diabetes is the greatest cause of blindness in younger people, and even con sidering all age groups is as common a cause of blindness as Glaucoma and Age Related Maculopathy. Diabetes causes a retinopathy (DR) which is basically a vasculopathy1,2 and the cellular biology of DR has been recently linked to cytokines.3 Starting from this fact, it has been proposed that techniques of molecular biology which might be of use in the treatment of inherited degenerative diseases could also be applicable to DR. This paper is however concerned with a far simpler method of controlling DR, which is based on the physiology of the eye, the particular features of which have been known for half a century but not exploited in this connection. The basic idea is that anoxia early in DR will only develop during dark adaptation, occuring in long periods every night in sleep. [ABSTRACT FROM AUTHOR]

Ribozymes targeted to a point mutation (histidine for proline substitution at position 23) in rhodopsin were evaluated as therapeutic agents for photoreceptor degeneration in an animal model of autosomal dominant retinitis pigmentosa (ADRP). Recombinant adeno-associated virus (AAV) vectors incorporating a proximal bovine rod opsin promoter were used to transfer either hairpin or hammerhead ribozyme genes to photoreceptors. AAV was administered by subretinal injection at postnatal day (P) 15 to rats expressing a rod opsin transgene with a proline-to-histidine substitution in exon 1 at codon 23 of the rhodopsin gene (P23H). In vivo levels of normal and mutant RNAs were measured by allele-specific RT-PCR at P60. Eyes were examined by histopathology and morphometric analysis, as well as by ERG at approximately P60, P75 or P90. Expression of either a hammerhead or hairpin ribozyme markedly slowed the rate of photoreceptor degeneration for at least 3 months as determined by outer nuclear layer thickness as well as by inner and outer segment length. Catalytically inactive control ribozymes had a significantly smaller effect on the retinal degeneration. Minimal rescue effects were seen in retinas injected with either AAV containing a gene for green fluorescent protein rather than the ribozyme gene or buffer only. The level of mutant opsin RNA relative to wild-type RNA was also reproducibly lower in ribozyme-treated retina. Finally, ERG comparisons of ribozyme-treated versus control eyes demonstrated functional rescue. We conclude that ribozyme-directed cleavage of mutant mRNAs appears to be a potentially effective therapy for autosomal dominant retinal degeneration. [ABSTRACT FROM AUTHOR]

Retinal support cells such as Müller cells and RPE are excellent candidates for targeting gene therapy vectors. However, the lack of knowledge of gene regulatory mechanisms in Müller cells has limited their use for designing cell type-specific expression vectors for gene therapy. As a first step in this direction, our recent research has focused on the identification of genetic regulatory elements for two Müller cell-specific genes—the glial intermediate filament protein (GFAP) gene and the cellular retinaldehyde-binding protein (CRALBP) gene. Experimental evidence from transfection assays suggest that promoter-proximal sequences are sufficient to drive expression of GFAP and CRALBP genes in Müller cells. However, studies with GFAP-transgenic mice suggest that GFAP gene transcription in Müller cells is complex, and that an inducible, Müller cell-specific enhancer is likely to control GFAP expression. Identification of the enhancer should provide a inducible, promoter system for expressing foreign proteins in the retina. [ABSTRACT FROM AUTHOR]

Ribozymes are RNA enzymes that can be modified to cleave almost any target RNA. Because of this versatility, they are useful in digesting transcripts of dominant mutant genes leading to retinal disease, including ADRP. One approach to gene therapy for this disease would be to reduce the expression of the mutant opsin that interferes with the accumulation of active rhodopsin and is responsible for a substantial fraction of ADRP. We have developed ribozymes designed to cleave mutant mRNA molecules leading to several opsin missense and nonsense mutations that cause retinal degeneration in human and in animal models. These include the P23H, G90D, S334 termination, and the P347S mutations. Both hairpin and hammerhead ribozymes have been tested. The efficiency of cleavage depends on the nucleotide triplet at the cleavage site and its context in the mRNA. All ribozymes tested are capable of cleaving short synthetic targets, longer cloned targets and full length opsin mRNA containing the mutant target sequence. In no case has cleavage of the wild-type opsin been detected. For these reasons, ribozymes hold promise as sequence-specific tools for gene therapy for ADRP. Animal tests of these ribozymes are underway using Adeno-Associated Virus vectors to deliver DNA copies of the ribozymes to affected transgenic animals. [ABSTRACT FROM AUTHOR]

Several Authors developed an evaluation of intraocular pressure (IOP) in normal population and in different groups of individuals, always measured with applanation tonometry that seems to be the most affordable and repeatible system. [ABSTRACT FROM AUTHOR]

From affinity purified SPACR we obtained seven peptides whose sequences reveal 100% identity to the deduced sequences in IMPG1 (accession number AF047492). The gene product of IMPG 1 is described as a chondroitin-6-sulfate proteoglycan. We report here that SPACR is the most prominent 147-150kD molecule present in the IPM. SPACR is not a chondroitin sulfate proteoglycan since it is not a product of chondroitinase ABC digestion and does not react with a highly specific chondroitin-6-sulfate antibody. Additionally, the deduced amino acid sequence of IMPG1 reveals no established glycosaminoglycan attachment site. We conclude that the glycoprotein SPACR is the gene product of IMPG1. SPACR is a heavily sialated glycoprotein containing both N- and O-linked glyconjugates. It is not the core protein of an IPM specific chondroitin sulfate proteoglycan. [ABSTRACT FROM AUTHOR]

Several studies demonstrated that genetically factors seem to influence the onset and progression of juvenile glaucoma as showed by different studies which identified linkage with chromosome #3q1,2,3 and #1g21-g31.4 Even if clearly is possible identify inherited factors in open angle chronic glaucoma (OACG) until now no candidate genes have been proposed for this disease. [ABSTRACT FROM AUTHOR]

Mutations in the gene encoding the disk rim specific protein, peripherin/rds, have been implicated in the pathogenesis of both autosomal dominant retinitis pigmentosa (ADRP) and various forms of macular dystrophy (MD). Peripherin/rds plays an important role in the morphogenesis and maintenance of the disk rim structure.This role is supported by interactions with other proteins. To better understand how these mutations lead to the development of disease, knowledge of the structure-function relationships of the peripherin/rds gene is needed.To facilitate these studies, genomic clones encoding the murine gene were isolated using bovine cDNA sequences as probes. Sequence analysis of clones that contained the entire coding sequence for mouse peripherin/rds revealed the exon-intron organization of the gene. The gene is composed of three exons (581, 247, and 213bp) and two introns. The first and second introns are 8.6 kb and 3.7 kb in size, respectively. Two major (1.6 and 2.7kb) and three minor (4.0, 5.5, 6.5kb) transcripts were detected on RNA blots. Several transcription start sites were mapped within 26 nucleotides located approximately 200bp upstream from the translation initiation site. However, the lengths of the 3'untranslated portion of the transcripts varied because of utilization of different polyadenylation signals. [ABSTRACT FROM AUTHOR]

Blue cone monochromacy (BCM) is an X-linked ocular disorder in which affected males have normal short-wavelength-sensitive (blue) cone and rod function but lack medium-(green) and long-wavelength-sensitive (red) cone function. Affected males characteristically have visual acuities of 20/100 to 20/200, myopia, nystagmus, and minimal foveal granularity with otherwise normal fundus findings.1 [ABSTRACT FROM AUTHOR]

Purpose: The goal of this study is to innovate a device for grading very low visual function expressed as light perception in patients such as retinitis pigmentosa (RP) and makes it possible to evaluate effects for various therapeutic modalities or their changes following the progress of the disease. Methods: The device was composed of a pair of goggles with a white light emission diode, 5 mm in diameter, an earphone for each ear, a control cabinet which emits light and a clicking stimuli, a hand-held grip with a button, and a recorder for calculating correct answer for stimuli. The light intensity and duration were changed in three levels, 10cd/m2(C), 100C, 1000C, and its duration, 0.1second(S), 0.3S, 1S independently. As a result, they were graded into nine different levels of intensities and delivered in a random sequence after a click sound of 0.7 sec prior to the stimulation. Patients were asked to push a button when they recognized these stimuli. Each eye was separately stimulated 5 times in each intensity and duration, 45 times per eye, therefore, a total of 90 times. Ten RP patients with light perception were examined and their visual ability was graded. The FP was examined before and after cataract extraction, stellate ganglion block and lipo-prostaglandin El was administered in two cases with RP and we studied the possibility of differentiating their functions and also reproducibility with this device. Results: We could differentiate changes of LP with this device and observe changes of very low grade vision with treatment. Conclusion: This device is valuable for evaluating low visual function such as LP of RP patients. It will be valuable for following their vision and also discriminating very small changes of visual function with medical and surgical treatment in the future. [ABSTRACT FROM AUTHOR]

Autosomal recessive retinitis pigmentosa (arRP) is a degenerative disease of photoreceptors in which defects in the genes encoding rhodopsin (RHO), α and β subunits of the cGMP phosphodiesterase (PDE6A and PDE6B, respectively), α subunit of the cGMP gated channel (CNCG), RPE65 and retinaldehyde-binding protein 1 (RLBP1) have been reported. Additionally, linkage analyses have defined four arRP loci on 1q31-q32 (RP12), 6p21.3 (RP14; mutations in the TULP1 gene), 1p13-p21 (RP19; mutation in the ABCR gene), and 2q31-33. However, the molecular basis of the disease has not been ascertained in over 50% of cases, yet. The PDE6A gene encodes the rod specific form of the a subunit of cGMP phosphodiesterase (PDE6). Together, theα and β subunits of cGMP PDE6 catalyze the breakdown of cGMP in response to light, a critical step in the light transduction pathway. The PDE6B gene has been analyzed in several arRP populations and it appears to account for less than 5% of all arRP cases. In contrast, fewer mutations have been reported in the PDE6A gene in arRP patients. Here we describe the genetic mapping of the PDE6A gene and the analysis of its involvement in arRP in 49 Spanish families. Linkage analysis performed in the CEPH panel of families allowed us to localize the gene at OcM from D5S434 (Z = 10.5). Cosegregation and homozygosity studies with an intragenic polymorphism, a very close marker (D5S434) and two flanking markers (D5S2013 and D5S436) ruled out PDE6A as the cause of arRP in 38 pedigrees. In the eleven remaining families, SSCP analysis of the PD E6A-coding region did not detect any disease causing mutation in the affected members. These results strongly suggest that mutations in the PDE6A gene are not responsible for arRP in these families. [ABSTRACT FROM AUTHOR]

Herein reported are the systemic and ophthalmologic features of four patients with Bardet-Biedl syndrome (BBS) from a consanguineous pedigree. Molecular genetic analyses showed linkage with the BBS4 locus (chromosome 15), and excluded linkage to the other known loci on chromosome 3 (BBS3), 11 (BBS1), and 16 (BBS2), respectively. Patients were 15 to 26 years old (M = 3; F = 1) at last examination, and followup was up to 10 years. An array of clinical and functional tests were performed to characterize the ocular phenotype of this pedigree. Mild obesity and dystrophic extremities were observed in all four subjects, and mild to moderate mental retardation was noted in three. All three male subjects had also cryptorchidism. Two siblings also had dental abnormalities. Renal function investigations were unrevealing. Night blindness was the presenting symptom in all patients (onset: birth to 4 years of age). Visual acuity and color vision were severely affected early on in three subjects, moderately in the fourth. Further deterioration was documented in the follow-up. On ophthalmoscopy, retinal vessels were severely attenuated in all cases. Retinal pigmentary changes were mild and sectoral in all subjects in the earlier phases of the disease. Optic discs were pale (partial to complete atrophy) in all patients. Increasing retinal pigmentation and disc pallor was documented over the years. Maculas appeared initially healthy in all subjects, but mild abnormalities became ophthalmoscopically apparent over time. Goldmann visual fields were variably constricted, with good preservation of the inferior field in one subject as late as 26 years of age. Maximal electroretinogram responses were recordable only in two subjects at first examination, and suggested possible partial preservation of rod function in the early phases of the retinopathy. Later in the course of the disease, only cone-mediated function could be identified. Despite the healthy appearance on ophthalmoscopy, macular cone function was profoundly altered in all subjects. In summary, the ophthalmologic manifestations of this BBS4 pedigree do not appear to be distinct from those of other reported BBS phenotypes. Systemic manifestations were only in part matching those described for other BBS4 kindreds, indicating substantial interfamilial heterogeneity. Particularly, obesity was not nearly as prominent a feature as reported for Arab-Bedouin BBS4 large consanguineous pedigrees. Intrafamilial variability was also noted, adding further to the complexity of this syndrome. [ABSTRACT FROM AUTHOR]

We are studying a large autosomal dominant family with a broad clinical spectum of macular degeneration. By analyzing microsatellite markers on chromosome 6, significant positive lod sores were obtained (D6S455: Z = 4.49 at theta = 0). Linkage analysis localizes the disease-causing gene to a 22cM interval on chromosome 6q11-13 and excludes the loci for North Carolina Macular Dystrophy and Progressive Bifocal Chorioretinal Atrophy. Three other retinalJ.macular dystrophies map to overlapping intervals and could be allelic forms of the same gene or from a cluster of genes causing retinal/macular degeneration. [ABSTRACT FROM AUTHOR]

Stargardt disease (STGD) and late-onset fundus flavimaculatus (FFM) are autosomal recessive conditions leading to macular degenerations in childhood and adulthood, respectively. Recently, mutations of the photoreceptor cell-specific ATP binding transporter gene (ABCR) have been reported in STGD on one hand, and in some age related macular dystrophies (AMD) on the other hand. In addition, autosomal recessive retinitis pigmentosa (RP19) has recently been ascribed to mutations in the same gene. To provide the first genotype-phenotype correlations in ABCR gene mutations, the screening of the 50 exons encoding ABCR have been performed in a large series of FFM and STGD families as well as in familial cases of AMD and in pedigrees segregating both STGD and RP19. Giving consideration to the results of this study, we suggest that homozygosity for truncating ABCR gene mutations result in severe RP while compound heterozygosity for frameshift and missense mutations at this locus result in a severe macular disorder, STGD. Conversely, all mutations identified in milder macular disorder, FFM, were missense mutations. Finally, some AMD patients were found to carry heterozygote ABCR gene mutations, suggesting that these mutations could represent in some cases a susceptibility factor for the disease. [ABSTRACT FROM AUTHOR]

Retinitis pigmentosa (RP) is a genetically heterogeneous group of diseases that show progressive degeneration of photoreceptors. Its incidence is about one in 3000 to 10,000 people in many countries, and it is a leading cause of blindness in the adult population. Several kinds of mutations have been reported in patients with autosomal recessive RP (arRP) in genes that are normally expressed in rod photoreceptors and that function in the process of phototransduction. Arrestin, one of the photoreceptorspecific proteins, plays a role in the cycle of rhodopsin regeneration. 1 A homozygous 1-base pair deletion mutation in the arrestin gene, designated the arrestin 1147delA, has been known as a frequent cause of Oguchi disease, a form of congenital stationary night blindness, in Japanese patients. 2 Interestingly, molecular genetic screening in patients with arRP, including a sibling of a patient with Oguchi disease, to search for mutations in the arrestin gene revealed that the same 1147delA mutation is also associated with arRP and is cosegregated with the disease. We report on 3 patients with arRP associated with the homozygous 1147delA mutation in the arrestin gene as evidence of variable expressivity of the mutation in the arrestin gene. [ABSTRACT FROM AUTHOR]

Arrestin is a 45 KDa protein and is mainly located in the membrane of photoreceptor rod cells and pineal gland. It is well known that 1147delA nutation in the arrestin gene causes Oguchi disease and autosomal recessive retinitis pigmentosa in Japanese patients. We examined expression of arrestin in mRNA isolated from whole blood cells of patients with Oguchi disease, who had the 1147delA mutation in the arrestin gene. Arrestin was expressed at the level of mRNA in all of three patients with Oguchi disease. mRNA from Oguchi patients did not have the same 1147delA mutation as was seen in the arrestin gene but had the normal sequence. The result indicated the possibility of RNA editing in blood cells in our patients. [ABSTRACT FROM AUTHOR]

In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the trans- Golgi network (TGN) upon expression in polarized epithelial Madin-Darby canine kidney (MDCK) cells. Truncated rhodopsin with a deletion of 32 C-terminal residues shows a non-polar steady-state distribution. Addition of the C-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin's cytoplasmic tail to a cytosolic protein glutathione 5-transferase (GST) also targets this fusion protein (GST-Rho39Tr) to the apical membrane. We conclude that the carboxy-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This result is in agreement with previous studies showing that the cytoplasmic tail of rhodopsin mediates its vectorial transport from its site of synthesis in the rod photoreceptor cell body to the rod outer segment, where phototransduction occurs. Several mutant rhodopsins, found in patients with autosomal dominant retinitis pigmentosa (ADRP), have an amino acid change at the carboxy-terminus and these mutants are defective in their outer segment localization. It will be of interest to examine the relationship between the defects in rhodopsin's targetingJ.transport and the pathogenesis of retinal degeneration. [ABSTRACT FROM AUTHOR]

Several mutations that cause severe forms of autosomal dominant retinitis pigmentosa (ADRP) cluster in the C-terminal region of rhodopsin. Recent studies have implicated the C-terminal domain of rhodopsin in its trafficking on specialized post-Golgi membranes to the rod outer segment (ROS) of the photoreceptor cell. In addition, aberrant subcellular localization of rhodopsin has been observed in transgenic animals carrying C-terminal mutations. To test if sequence within the C-terminal domain regulates rhodopsin sorting to the ROS, we tested the effects of synthetic peptides that mimic this domain on intracellular trafficking of rhodopsin reconstituted in the frog retinal cell free system. The rhodopsin C-terminal sequence QVS(A)PA is highly conserved among different species. Synthetic peptides corresponding to the C-terminal of frog (AA 330-354) and bovine (AA 324-348) rhodopsin inhibited post-Golgi trafficking by 60% and 50%, respectively, and arrested newly synthesized rhodopsin in the trans-Golgi network (TGN). Peptides corresponding to the cytoplasmic loops and several control peptides had no effect. To assess the role of the last 5 amino acids QVS(A)PA in rhodopsin trafficking, and to model three naturally occurring mutations: Q344ter (lacking the last 5 amino acids QVAPA), V345M and P347S, we introduced equivalent substitutions into the frog C-terminal peptide. Each of these substitutions completely abolished the inhibitory activity of the peptides. Our data could help to explain detrimental effect of mutant rhodopsin in patients with ADRP. We propose that amino acids QVS(A)PA comprise a sorting signal that is recognized by specific factor(s) in the trans-Golgi network. A lack of recognition of this sequence, due to mutations in the last five amino acids causing ADRP most likely results in abnormal post-Golgi membrane formation and in an aberrant subcellular localization of rhodopsin. [ABSTRACT FROM AUTHOR]

The rod opsin Pro23His mutation accounts for approximately 12% of autosomal dominant retinitis pigmentosa (adRP) cases in the United States. To study mechanisms leading to photoreceptor degeneration, we investigated the effects of mutant and wild-type opsin stoichiometry on retinal morphology and function. We crossbred a trans-genic mouse line expressing the triple mutant, V20G, P23H, and P27L (GHL), with rhodopsin knockout mice. Retinal morphology of 30-day old GHL+ mice with two functional copies of the rod opsin gene (GHL+, rho+/+), one functional copy (GHL+, rho+/−) or no functional copies of the rod opsin gene (GHL+, rho−/−) was examined. Although mice of all three genotypes underwent reunal degeneration, the severity of the retinopathy correleted inversely with the number of wild-type opsin genes present. Mice with no functional wild-type opsin gene were most severely affected, while those with two functional copies were the least affected. Mice with a single functional vvild-type gene were intermediate in the degenerative phenotype. Correspondingly, changes in fundus morphology and ERG function. were most prominent in GHL+, rho−/− mice, whereas GHL+, rho−/− mice were similar to wild-type mice. [ABSTRACT FROM AUTHOR]

Purpose: Clinical evaluation of 42 patients with primarily suspected diagnosis of Leber's congenital amaurosis (LCA). Methods: We used the electroretinogram (ERG), visual acuity, fundus ap- pearance, age of onset, and systemic findings to confirm or revise the diagnosis of LCA. Results: Twenty-four patients had a non-recordable ERG, and 3 showed small, residual, mainly cone-driven b-waves. Profound visual loss ranged from no light perception to 20/200. Visual acuity of light perception was frequent at <1 year of age. Three patients never had light perception. Most patients had a stable visual acuity despite progressive retinal pigmentary changes. A normal fundus was seen predominantly in patients younger than 1 year of age, but could also be found in patients of all age groups. The first, and frequently the only pigmentary changes were vascular attenuation and rarefaction, later in life heterogeneous retinal findings appeared. None of the 9 reexamined patients showed a normal fundus. Nystagmus and strabismus were principal presenting symptoms. Eleven patients presented with the oculodigital sign, three of them were enophthalmic, and 12 patients showed roving eye movements. Patients with additional (nonocular) findings such as mental retardation (n = 12), renal (n = 3) and skeletal (n = 4) abnormalities revealed no different ERG- or ophthalmoscopic findings than the remaining patients. Twelve patients revealed abnormal psychomotor development. From the initial cohort of 42 patients with suspected LCA, the diagnosis had to be revised in 15 cases as follows: juvenile RP (n = 8), infantile Refsum syndrome (n = 1), and 6 patients could not yet be classified with certainty. Conclusion: Bilateral visual impairment in infants should be assessed clinically and electroretinographically within the first year. Later in life, the diagnosis of LCA can be a challenge due to its phenotypical heterogeneity. Systemic disorders are inde pendent of ocular findings. In patients older than two years with non-recordable ERG, visual acuity better than 201200, progressive visual loss, normal pupillary responses, absent oculodigital sign and absence of systemic abnormalities, juvenile retinitis pigmentosa should be considered. [ABSTRACT FROM AUTHOR]

The first description of the retinal degeneration (rd) chicken appeared in the literature in 1980. From the early 1980s to 1991, Ulshafer and his colleagues carried out detailed electrophysiological, morphological, and biochemical analyses of the retinas of these animals, the results of which provided a comprehensive description of the pathology of this retinal degenerative disease, and the first clues concerning the identity of the rd gene. In 1990, we embarked on a series of molecular studies that would eventually lead to the identification of the rd gene in 1998. As a result of the tireless efforts of many individuals, the rd chicken is now recognized as a model for Leber congenital amaurosis, Type 1, a devastating human disease that causes blindness in newborn infants. With the identity of the rd gene in hand, it seems appropriate to review the nearly two decades of research that has been conducted on this model, research that has provided a firm foundation for our current efforts to restore sight in these animals using somatic gene therapy. [ABSTRACT FROM AUTHOR]

Guanylyl cyclase-E (GC-E), which is specifically expressed in the retina, has been proposed as critical for normal vision. Mutations within the human homolog of the rodent GC-E gene have been reported in patients with Leber congenital amaurosis (LCA) and in patients with cone-rod dystrophy (CORD6). We have recently produced a mouse genetic model lacking the GC-E gene, and have recorded electroretinograms (ERGs) from mice at different ages. Rod ERGs to flashes over a 6 log unit range were recorded from 18 GC-E −/−, 4 GC-E +/−, and 30 GC-E +/+ mice. Derived parameters were used to assess activation and inactivation stages of transduction, inner retinal function, and cone system activity. GC-E +/− mice were indistinguishable from normal in all measures. In GC-E −/− mice, the most striking abnormality was a severe reduction in the cone ERG by 3-5 wks of age and a complete absence of a detectable cone ERG in mice >8 wks. An early loss of cones or a failure of cones to develop was confirmed by histological analysis. The maximum rod photoresponse amplitude (Rm) was 70% lower than normal at 3-5 wks despite a normal histological appearance of rods. Recovery from activation, as assessed by a double-flash technique, was more rapid in GC-E −/− than in wild-type mice. Reductions in b-wave amplitude at all ages were accompanied by a shortening of b-wave implicit time. These findings suggest that GC-E is critical for the survival of cone, but not rod, photoreceptors. [ABSTRACT FROM AUTHOR]