Photoreceptor cell rescue in retinal degeneration (rd) mice by in vivo gene therapy.

Mutations in the beta subunit of the cGMP phosphodiesterase gene (beta PDE) can cause a recessively inherited retinal degeneration in several species, including mice, dogs and humans. We tested the possibility of altering the course of retinal degeneration in the rd mouse through subretinal injection of a recombinant replication-defective adenovirus that contains the murine cDNA for wild-type (beta PDE, Ad.CMV beta PDE. Subretinal injection of Ad.CMV beta PDE results in beta PDE transcripts and increased PDE activity and delays photoreceptor cell death by six weeks. The findings demonstrate cell rescue by in vivo gene transfer, thus supporting the feasibility of treating an inherited retinal degeneration by somatic gene therapy.

Trichromatic mechanisms in single cortical neurons.

By chromatic adaptation, all three cone mechanisms of rhesus monkey vision can be identified in single neurons of striate cortex. This trichromatic inter-action occurs in cells sensitive to color and indicates that striate cortical cells tend to be more wavelength discriminating than cells at lower stages of the primate visual system.

Enchancement of luminance flicker by color-opponent mechanisms.

Color-opponent ganglion cells in the monkey retina respond to luminance flicker at high temporal frequencies. Color opponency, which makes these cells so selective of wavelength at low temporal frequencies, is progressively lost at high frequencies. This loss is due to a frequency-dependent phase shift between the responses of spectrally different center and surround mechanisms in the receptive field of each of these cells. Center and surround responses, which are antagonistic at low temporal frequencies, become synergistic at high ones, making these cells most responsive at high frequencies to those wavelengths to which they are least responsive at low frequencies. This phenomenon can explain the differences between chromatic and luminance flicker in human vision.

Horizontal cells in cat retina with independent dendritic systems.

Cat horizontal cells are retinal neurons with two functionally distinct parts: the cell body receives signals predominantly from cones, while the terminal arborization receives predominantly from rods. The long thin process connecting these parts neither generates impulses nor allows significant passive electrotonic conduction between them.

Retinal degeneration in mice lacking the gamma subunit of the rod cGMP phosphodiesterase.

The retinal cyclic guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) is a key regulator of phototransduction in the vertebrate visual system. PDE consists of a catalytic core of alpha and beta subunits associated with two inhibitory gamma subunits. A gene-targeting approach was used to disrupt the mouse PDEgamma gene. This mutation resulted in a rapid retinal degeneration resembling human retinitis pigmentosa. In homozygous mutant mice, reduced rather than increased PDE activity was apparent; the PDEalphabeta dimer was formed but lacked hydrolytic activity. Thus, the inhibitory gamma subunit appears to be necessary for integrity of the photoreceptors and expression of PDE activity in vivo.

Role for the target enzyme in deactivation of photoreceptor G protein in vivo.

Heterotrimeric guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are deactivated by hydrolysis of the GTP that they bind when activated by transmembrane receptors. Transducin, the G protein that relays visual excitation from rhodopsin to the cyclic guanosine 3',5'-monophosphate phosphodiesterase (PDE) in retinal photoreceptors, must be deactivated for the light response to recover. A point mutation in the gamma subunit of PDE impaired transducin-PDE interactions and slowed the recovery rate of the flash response in transgenic mouse rods. These results indicate that the normal deactivation of transducin in vivo requires the G protein to interact with its target enzyme.

Bestrophin detected in the basal membrane of the retinal epithelium and drusen of monkeys with drusenoid maculopathy.

PURPOSE: To determine if bestrophin is present in the basal membrane of macular retinal pigment epithelium (RPE) and in drusen of rhesus monkeys with age-related drusenoid maculopathy. METHODS: The macular region of three rhesus monkeys (Macaca mulatta), 23-24 years of age, with drusenoid maculopathy was dissected from eyes fixed with 4% paraformaldehyde. The macula was sectioned into rectangular pieces. The sclera was removed from each segment and the remainder separated into segments of neural retina with retinal epithelium or choroid with retinal epithelium. These segments were incubated with a goat polyclonal antibody to human bestrophin 1, reacted with gold-labeled rabbit antibody to goat IgG, silver-enhanced, and processed for transmission electron microscopy. RESULTS: Bestrophin-labeled gold particles were found in quasi-linear arrays on the basal surface of the macular RPE and also within drusen where bestrophin was found in segments of membranous-like material. The array density of the bestrophin-linked gold particles on the basal membrane of the epithelium had a maximal value of about 5-100 bestrophin molecules/micron(2). Immuno-detection of bestrophin was most effective when examined in an RPE layer that remained attached to the neural retina, where the basal surface of the epithelium is more directly exposed to the antibodies. CONCLUSION: Bestrophin is present on the basal membrane of macular RPE of rhesus monkeys with age-related drusenoid maculopathy, and also found in the membranous-like structures of drusen. The latter finding provides insight into the pathogenesis of drusen by indicating that segments of the basal membrane of RPE contribute to the material that accumulates within drusen.

Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy.

Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The disease phenotype that is most recognized in STGD1 patients, and also in the Abca4-/- mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. Here, we tested whether delivery of the normal (wt) human ABCA4 gene to the subretinal space of the Abca4 -/- mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the lipofuscin pigment A2E. Equine infectious anemia virus (EIAV)-derived lentiviral vectors were constructed expressing either the human ABCA4 gene or the LacZ reporter gene under the control of the constitutive (CMV) or photoreceptor-specific (Rho) promoters. Abca4-/- mice were injected subretinally with 1 microl ( approximately 5.0 x 10(5) TU) of each EIAV vector in one eye at postnatal days 4 and 5. An injection of saline, an EIAV-null vector, or an uninjected contralateral eye served as a control. Mice were killed at various times after injection to determine photoreceptor (PR) transduction efficiency and A2E concentrations. EIAV-LacZ vectors transduced from 5 to 20% of the PRs in the injected area in mice. Most importantly, a single subretinal injection of EIAV-CMV-ABCA4 to Abca4-/- mouse eyes substantially reduced disease-associated A2E accumulation compared to untreated and mock-treated control eyes. Treated eyes of Abca4-/- mice accumulated 8-12 pmol per eye (s.d.=2.7) of A2E 1 year after treatment, amounts comparable to wt controls, whereas mock-treated or untreated eyes had 3-5 times more A2E (27-39 pmol per eye, s.d.=1.5; P=0.001-0.005). Although extrapolation to humans requires caution, the high transduction efficiency of both rod and cone photoreceptors and the statistically significant reduction of A2E accumulation in the mouse model of STGD1 suggest that lentiviral gene therapy is a potentially efficient tool for treating ABCA4-associated diseases.

Novel organelles in primate retinal epithelium.

We are investigating age-related changes in organelles in monkey retinal epithelium using transmission and analytic electron microscopy. We previously described a circular organelle in retinal epithelium with a diameter of about 0.5µm. The organelle is unique in containing a single, round vacuole within an otherwise electron dense interior. We suggested that the organelle might be a melanosome with lysosomal properties. We now find that there are two similar organelles with such a single vacuole but which differ in their chemical composition, electron density, cell location and according to age. Epon embedded sections from the macular epithelium of seven monkeys, ranging from 1 to 35 years of age, were examined by transmission electron microscopy. A seven year old monkey was processed for analytic electron microscopy to determine the chemical composition of the organelles. The number and location of the organelles in the retinal epithelium were determined. The chemical composition of these two organelles was different. One of the organelles contained high mole fractions of oxygen and nitrogen and little phosphorous characteristic of melanin; the other had little oxygen and nitrogen and higher mole fractions of phosphorous uncharacteristic of melanin, but more common with lysosomal organelles. The latter had an electron dense rim around the vacuole, a less electron dense interior than the melanin containing organelle and also contained iron. The melanin containing organelle was more common in young monkeys and in the middle third of the cell. The organelle without melanin was more common in old monkeys and localized in the basal third of the cell. Two similarly vacuolated organelles, not identified before in retinal epithelium, differ in their chemical composition. One contains melanin; the other does not. The former is more common in young and the latter more common in old monkeys. This suggests reorganization and or degradation of melanin-containing organelles with age. These changes show how analytic electron microscopy can distinguish major ultra-structural differences in organelles when mere observation fails to do so easily.

Retroviral gene transfer into retinal pigment epithelial cells followed by transplantation into rat retina.

In this preliminary report, we describe a technique for gene transfer into the retina using a retrovirus vector. We transferred the bacterial LacZ gene and the neomycin-resistance gene into pigmented wild-type rat retinal pigment epithelial (RPE) cells in culture. The RPE culture was exposed to retrovirus, and infected cells were selected with a neomycin analog (G418). The LacZ gene product was detected by X-Gal histochemistry in 95-100% of drug-resistant cells. These genetically labeled cells were transplanted into the subretinal space of two 15- to 25-day-old albino RCS rats, which have an inherited retinal degeneration syndrome. The retinas were fixed and stained with X-Gal at 3 and 6 weeks after transplantation. At both time points, pigmented, LacZ-containing cells were seen in the subretinal space. Further, there were several rows of photoreceptor nuclei in the transplant area of the approximately 2-month-old rats, while in the control contralateral eye the photoreceptor nuclei were virtually absent, as for untreated animals, suggesting that the transplanted LacZ-marked, wild-type RPE cells may have helped preserve photoreceptors. The technique for gene transfer into RPEs followed by transplantation thus provides a means for gene therapy in organisms with a genetic defect in RPE cells.

Genotyping microarray (gene chip) for the ABCR (ABCA4) gene.

Genetic variation in the ABCR (ABCA4) gene has been associated with five distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), and age-related macular degeneration (AMD). Comparative genetic analyses of ABCR variation and diagnostics have been complicated by substantial allelic heterogeneity and by differences in screening methods. To overcome these limitations, we designed a genotyping microarray (gene chip) for ABCR that includes all approximately 400 disease-associated and other variants currently described, enabling simultaneous detection of all known ABCR variants. The ABCR genotyping microarray (the ABCR400 chip) was constructed by the arrayed primer extension (APEX) technology. Each sequence change in ABCR was included on the chip by synthesis and application of sequence-specific oligonucleotides. We validated the chip by screening 136 confirmed STGD patients and 96 healthy controls, each of whom we had analyzed previously by single strand conformation polymorphism (SSCP) technology and/or heteroduplex analysis. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded many sequence changes undetected by SSCP. In STGD patient cohorts, the efficiency of the array to detect disease-associated alleles was between 54% and 78%, depending on the ethnic composition and degree of clinical and molecular characterization of a cohort. In addition, chip analysis suggested a high carrier frequency (up to 1:10) of ABCR variants in the general population. The ABCR genotyping microarray is a robust, cost-effective, and comprehensive screening tool for variation in one gene in which mutations are responsible for a substantial fraction of retinal disease. The ABCR chip is a prototype for the next generation of screening and diagnostic tools in ophthalmic genetics, bridging clinical and scientific research.

Uniqueness of the S-cone pedicle in the human retina and consequences for color processing.

The purpose of this study was to investigate more fully the shape and content of ribbons and synapses to second-order neurons in the short-wavelength cone (S-cone, blue cone) pedicle and to learn more concerning the uniqueness of the S-cone system in the primate retina. A piece of well-fixed peripheral human retina (10 mm, 35 degrees nasal to the fovea) was serially thick sectioned in the tangential plane from the level of the outer segments to the tops of the cone pedicles. Then serial electron microscope (EM) sections were collected through the whole depth of the pedicle-occupying region into the neuropil of the outer plexiform layer (OPL). The resultant EM micrograph montages of a large field of cone pedicles were perused, and S-cone pedicles were identified. Serial micrographs of a single S-cone pedicle, picked out of the montages, were digitized and reconstructed by computer three-dimensional methods. The S-cone pedicle arose from a slightly oblique axon and projected 0.5-1 microm more vitread in the OPL than other cone pedicles. It was bilobed in shape, with synaptic invaginations and ribbons in both lobes. No cone-contacting telodendria projected from the S-cone pedicle itself, but a small number of neighboring cones sent telodendria to its surface to make small gap junctions. Neighboring rod spherules also made small gap junctions. Four robust bipolar cell dendrites, most likely from S-cone-specific bipolar cells, made synapses at ribbons and basal (distal) junctions. A small number of other bipolar cell dendrites made narrow-cleft basal junction only. The majority of lateral elements were thought to be from HII horizontal cells, and a minority from HI horizontal cells. We conclude that the S-cone pedicle has a unique morphology and connectivity to second-order neurons that makes it quite different from the other two longer wavelength cone systems, and we speculate on the consequences for color processing in the visual system in general.

Electrophysiologic features of abetalipoproteinemia: functional consequences of vitamin E deficiency.

We performed electrophysiologic testing in 10 patients with abetalipoproteinemia (ABL). Peripheral nerve studies implied an axonal disorder. Visual evoked potentials demonstrated prolonged P100 latency in three patients and abnormal electroretinograms in six. Somatosensory evoked potentials indicated dorsal column dysfunction in eight patients. Brainstem auditory evoked potentials were normal. Findings were consistent with the known neuropathology of ABL and of experimental vitamin E deficiency. Stabilization or improvement in electrophysiologic findings occurred with vitamin E supplementation. Neurophysiologic tests document retinal, central somatosensory and peripheral nerve lesions in vitamin E deficiency and provide an objective indication of response to treatment.

Blue-sensitive cones of the cat produce a rodlike electroretinogram.

Two cone mechanisms are identifiable in the strongly yellow light-adapted electroretinogram (ERG) of the arterially perfused cat eye. One has its maximum spectral sensitivity near 555 nm; the other has its maximum near 450 nm. The former cone system produces a much larger signal with characteristics of a typical cone or inhibitory ERG. The latter cone system produces a small, saturable signal (less than 5 microV) which resembles a rodlike or excitatory ERC. The results imply that the latter ERG is generated by blue-sensitive cones, which form a small fraction of the total cone population and share some physiological and perhaps anatomical properties of rods.

Electroretinographic responses of the short-wavelength-sensitive cones.

An electroretinographic response of the human short wavelength (S) cone system can be distinguished from that of the longer wavelength (L or M) cone system by using ganzfeld short-wavelength stimulation at relatively high levels of retinal adaptation. The S cone response has both an a- and b-wave component in its ERG, both of which are slower than those of the L or M cone response at the same level of retinal adaptation. Proof that this is the S cone response is obtained by action spectra and by examination of a sex-linked achromat who is known to have only S cone and rod vision. This approach allows the simultaneous and rapid assessment of both the S and the L or M cone systems in the human retina using conventional electroretinogram (ERG) equipment, ganzfeld blue flashes on a white background, and computer averaging.

Light adaptation of the electroretinogram. Diminished in retinitis pigmentosa.

The cone electroretinogram has been examined at different levels of ganzfeld adaptation in normals and subjects with retinitis pigmentosa (RP). As light adaptation increases, the amplitude and time course (measured as b-wave implicit time) of the cone ERG decrease in both normal and RP subjects. The same level of light adaptation, however, decreases the amplitude and the implicit time more in normal than in RP subjects. The ineffectiveness of light for adapting the ERG of RP subjects is most easily explained by assuming that RP cones absorb less light than normal cones. By comparing these parameters between normal and RP subjects at different levels of light adaptation, it is possible to estimate this ineffectiveness of cone absorption in RP subjects. The results imply that RP cones can transduce and adapt but fail to absorb light as effectively as normal cones. The quantitative relationship between cone b-wave implicit time and retinal illumination provides a unique method for examining cone function as well for standardizing ganzfeld backgrounds in ERG laboratories.

Growth in amplitude of the human cone electroretinogram with light adaptation.

The human cone electroretinogram gradually increases in amplitude an average of 75% (range 23 to 157%) during light adaptation, over a period of approximately 20 min. This increase involves both the a- and b-wave components of this response, and both waves follow a similar time course, implying that the photoreceptors themselves are responsible for the effect. The phenomenon occurs with suprathreshold, but not with threshold, levels of stimulation, and the stronger the test light, the greater the effect. An increase in the intensity of the adapting light shortens the time course of the ERG response, measured as b-wave implicit time, but this occurs almost immediately, and the implicit time then remains constant during the slow increase in response amplitude. The stronger the background adapting light, the smaller is the ERG amplitude, but the percentage growth (or rate of recovery) is unchanged. This slow increase in amplitude is thought to reflect the redepolarization of the cones, after their initial hyperpolarization to an adapting field. It does not reflect the d.c. potential of the eye (the EOG). It is essential to control this phenomenon in any studies of the human cone ERG, in order to minimize variability.

Supernormal cone electroretinograms in central retinal vein occlusion.

In 12 successive cases of unilateral central retinal vein occlusion (CRVO), the strongly light-adapted cone electroretinogram (both a- and b-wave) was always slower and larger (supernormal) to long-wave stimuli compared with that of the unaffected eye. This supernormality became less as the level of light adaptation decreased; in the dark-adapted state, long-wave stimuli produced subnormal responses from the affected eye in all but two subjects. This supernormality was not caused by ineffectiveness of the adapting light related to a reduced cone quantal catch because it occurred in the dark. At any one state of adaptation, the supernormality increased with the wavelength of stimulation, paralleling the relative absorption ratio of long-middle wavelength-sensitive cones. This suggests that cones, especially long wavelength-sensitive cones, are less able to reduce their responsiveness to light with increasing levels of light adaptation in a retina affected by CRVO.

The human S-cone electroretinogram and its variation among subjects with and without L and M-cone function.

PURPOSE: To examine the S-cone ERG in subjects with and without L and M-cone function. METHODS: Ganzfeld spectral flashes in the presence of strong Ganzfeld adapting fields are used to elicit S-cone ERGs. RESULTS: The S-cone ERG b-wave ranges from 0.2 to 4 mV in amplitude and 38-45 msec in implicit time. There is a progressive decrease in amplitude with age. The response is similar in subjects with or without L and M cone function. CONCLUSION: The S-cone ERG is detectable in subjects of all ages, but intersubject variability limits its diagnostic usefulness. The S-cone ERG is slightly later than but does not appear to be obviously influenced by the L and M-cone ERG.

Retinal pigment epithelial transplants and retinal function in RCS rats.

PURPOSE: To determine if retinal pigment epithelium (RPE) transplantation maintains visual function in Royal College of Surgeons (RCS) strain of rats. METHODS: Twelve RCS rats received RPE transplants at 16 to 20 days after birth. The retinas were studied electrophysiologically and histologically from 3 to 10 months after transplantation and compared with 11 RCS controls and 11 normal rats of comparable ages. A microelectrode was guided to the transplant site visible by its pigmentation in the albinotic RCS retina to detect responses. RESULTS: Spontaneous ganglion cell activity was present in all retinas. Ganglion cell responses to light were detected in 9 of the 12 transplant eyes but not in any of the 11 controls. 96, 44, 140 units were encountered and 30%, 0%, 97% were driven by light respectively in transplant, control, and normal retinas. In transplants 36%, 29%, and 28% were driven at 3 to 4, 6 to 7, and 10 months after transplantation, respectively. Intraretinal ERGs with both a- and b-waves were recorded in 5 of the 8 transplants studied. None of the RCS controls studied had an IERG. The average IERG was 2.5 microV (SD = 1.9) in transplants and 59 microV (SD = 19) in normal retinas. The electrode track was traced to the transplant site in six of the seven retinas that were responsive to light and examined histologically. CONCLUSION: RPE transplants to RCS rats maintain retinal function in the transplant site for long periods of time.