Muayyad R. Al-Ubaidi

Cloning and Expression of Two Related Connexins from the Perch Retina Define a Distinct Subgroup of the Connexin Family

We have cloned cDNAs for two closely related connexins (Cx), Cx35 and Cx34.7, from a perch retinal cDNA library. Sequencing of PCR products from genomic DNA revealed that both connexins have an intron 71 bp after the translation initiation site; in Cx35, the intron is 900 bp in length, whereas in Cx34.7 it is ∼20 kb. Southern blots of genomic DNA suggest that the two connexins represent independent single copy genes. In Northern blots, Cx35 and Cx34.7 transcripts were detected in retina and brain; Cx34.7 also showed a weak signal in smooth muscle (gut) RNA. Antibodies against Cx35 labeled a 30 kDa band on a Western blot of retinal membranes, and in histological sections, the pattern of antibody recognition was consistent with labeling of bipolar cells and unidentified processes in the inner plexiform and nerve fiber layers. When expressed in Xenopus oocytes, Cx35 and Cx34.7 formed homotypic gap junctions, but the junctional conductance between paired oocytes expressing Cx35 was 10-fold greater than that recorded for gap junctional channels formed by Cx34.7. The homotypic gap-junctional channels were closed in a voltage-dependent manner but with relatively weak voltage sensitivity. Heterotypic gap junctions formed by Cx35 and Cx34.7 displayed junctional conductances similar to those of Cx34.7 homotypic pairs and showed a slightly asymmetric current–voltage relationship; the side expressing Cx35 exhibited a higher sensitivity to transjunctional potentials. An analysis of the sequence and gene structure of the connexin family revealed that perch Cx35 and Cx34.7, skate Cx35, and mouse Cx36 constitute a novel γ subgroup.

Properties of the mouse cone-mediated electroretinogram during light adaptation

Cone-mediated electroretinograms (ERGs) were obtained from normal mice during the course of light adaptation to a rod-desensitizing adapting field. Responses obtained during the early minutes of light adaptation were smaller in amplitude, and delayed in implicit time in comparison to responses obtained to the same stimulus presented later during light adaptation. These changes resemble those observed in the human cone ERG obtained under similar stimulus conditions, and indicate that the underlying mechanism may be similar. While the use of an adapting field appears to isolate the mouse cone ERG, these adaptation-induced changes should be considered when evaluating this response.

Retina-derived microglial cells induce photoreceptor cell death in vitro

In animals with retinal degeneration, the presence of activated microglial cells in the outer retina during the early stages of injury suggests that they may be involved in the ensuing photoreceptor cell death. In the following study, we investigated the effects of rat retina-derived microglial cells on a photoreceptor cell line (661w) using cell culture techniques. The difficulty of obtaining pure populations of photoreceptor cells necessitated our use of the 661w photoreceptor cells generated from retinas of transgenic mice. 661w Cells were incubated for 24-48 h in basal medium or basal medium conditioned by activated microglial cells (MGCM) or Müller cells (MCCM), and tested for cell death using lactate dehydrogenase (LDH) assay. The induction of apoptosis in the 661w cells by MGCM was investigated using Terminal deoxynucleotidyl Transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and DNA laddering. Treatment of 661w cells with MGCM for 48 h resulted in approximately 73% of cells dead as compared with 19-20% of cells grown in either basal medium or MCCM. Serum supplementation or pretreatment with heat did not abolish the cytotoxicity of MGCM. More TUNEL-positive cells were observed in MGCM-treated cultures as compared with those in basal medium. Bands in multiples of approximately 180 bp formed DNA ladders in MGCM-treated but not in basal medium-treated samples. Our study shows that microglial cells release soluble product(s) that induce degeneration of cultured photoreceptor cells. Moreover, the mechanism of microglia-induced photoreceptor cell death may involve apoptosis similar to that observed in animals with retinal degeneration.

Functional characteristics of skate connexin35, a member of the γ subfamily of connexins expressed in the vertebrate retina

Retinal neurons are coupled by electrical synapses that have been studied extensively in situ and in isolated cell pairs. Although many unique gating properties have been identified, the connexin composition of retinal gap junctions is not well defined. We have functionally characterized connexin35 (Cx35), a recently cloned connexin belonging to the γ subgroup expressed in the skate retina, and compared its biophysical properties with those obtained from electrically coupled retinal cells. Injection of Cx35 RNA into pairs of Xenopus oocytes induced intercellular conductances that were voltage‐gated at transjunctional potentials ≥ 60 mV, and that were also closed by intracellular acidification. In contrast, Cx35 was unable to functionally interact with rodent connexins from the α or β subfamilies. Voltage‐activated hemichannel currents were also observed in single oocytes expressing Cx35, and superfusing these oocytes with medium containing 100 μm quinine resulted in a 1.8‐fold increase in the magnitude of the outward currents, but did not change the threshold of voltage activation (membrane potential = +20 mV). Cx35 intercellular channels between paired oocytes were insensitive to quinine treatment. Both hemichannel activity and its modulation by quinine were seen previously in recordings from isolated skate horizontal cells. Voltage‐activated currents of Cx46 hemichannels were also enhanced 1.6‐fold following quinine treatment, whereas Cx43‐injected oocytes showed no hemichannel activity in the presence, or absence, of quinine. Although the cellular localization of Cx35 is unknown, the functional characteristics of Cx35 in Xenopus oocytes are consistent with the hemichannel and intercellular channel properties of skate horizontal cells.

Overexpression of Rhodopsin Alters the Structure and Photoresponse of Rod Photoreceptors

Rhodopsins are densely packed in rod outer-segment membranes to maximize photon absorption, but this arrangement interferes with transducin activation by restricting the mobility of both proteins. We attempted to explore this phenomenon in transgenic mice that overexpressed rhodopsin in their rods. Photon capture was improved, and, for a given number of photoisomerizations, bright-flash responses rose more gradually with a reduction in amplification--but not because rhodopsins were more tightly packed in the membrane. Instead, rods increased their outer-segment diameters, accommodating the extra rhodopsins without changing the rhodopsin packing density. Because the expression of other phototransduction proteins did not increase, transducin and its effector phosphodiesterase were distributed over a larger surface area. That feature, as well as an increase in cytosolic volume, was responsible for delaying the onset of the photoresponse and for attenuating its amplification.

Degeneration of cone photoreceptors induced by expression of the Mas1 protooncogene.

Although transgenic expression of oncogenes typically leads to tumorigenesis, oncogene expression directed to the rod photoreceptors leads to cell death without tumor formation. To evaluate the cellular and functional changes induced in cone photoreceptors by an oncogene, the Mas1 protooncogene was targeted to the cones of transgenic mice by the human red/green opsin promoter. Mas1 was chosen because of its exclusive expression in the nervous system and its homology to opsin. The overall histologic appearance of the transgenic retina was normal and retinal tumors were never observed. While rod-mediated electroretinograms were normal in all respects, cone-mediated responses were diminished in direct relationship to the level of transgene expression as determined by Northern blot analysis. Responses of UV- and green-sensitive cones were reduced equivalently, and Northern analysis and immunocytochemistry indicated that cone photoreceptor densities were markedly diminished throughout transgenic retinas. These results indicate that oncogene expression in cones induces cell death without tumor formation and support the possibility that aberrant oncogene expression may underlie some forms of hereditary retinal diseases. The Mas1 transgenic mice may be useful in understanding the cone photoreceptor degeneration that occurs in cone dystrophies and age-related macular degeneration and in evaluating potential therapies for these disorders.

G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor.

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319–43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific α-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65-/- mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction.

Retinal abnormalities associated with the G90D mutation in opsin

Several mutations in the opsin gene have been associated with congenital stationary night blindness, considered to be a relatively nonprogressive disorder. In the present study, we examined the structural and functional changes induced by one of these mutations, i.e., substitution of aspartic acid for glycine at position 90 (G90D). Transgenic mice were created in which the ratio of transgenic opsin transcript to endogenous was 0.5:1, 1.7:1, or 2.5:1 and were studied via light and electron microscopy, immunocytochemistry, electroretinography (ERG), and spectrophotometry. Retinas with transgenic opsin levels equivalent to one endogenous allele (G0.5) appeared normal for a period of about 3–4 months, but at later ages there were disorganized, shortened rod outer segments (ROS), and a loss of photoreceptor nuclei. Higher levels of G90D opsin expression produced earlier signs of retinal degeneration and more severe disruption of photoreceptor morphology. Despite these adverse effects, the mutation had a positive effect on the retinas of rhodopsin knockout (R−/−) mice, whose visual cells fail to form ROS and rapidly degenerate. Incorporation of the transgene in the null background (G+/−/R−/− or G+/+/R−/−) led to the development of ROS containing G90D opsin and prolonged survival of photoreceptors. Absorbance spectra measured both in vitro and in situ showed a significant reduction of more than 90% in the amount of light‐sensitive pigment in the retinas of G+/+/R−/− mice, and ERG recordings revealed a >1 log unit loss in sensitivity. However, the histological appearances of the retinas of these mice show no significant loss of photoreceptors and little change in the lengths of their outer segments. These findings suggest that much of the ERG sensitivity loss derives from the reduced quantal absorption that results from a failure of G90D opsin to bind to its chromophore and form a normal complement of light‐sensitive visual pigment. J. Comp. Neurol. 478:149–163, 2004.

Functional consequences of oncogene-induced photoreceptor degeneration in transgenic mice.

This study evaluated retinal function in mice following the expression of oncogenes under the control of photoreceptor-specific promoters in transgenic mice. Electroretinograms (ERGs) were recorded under stimulus conditions chosen to elicit rod- or cone-mediated components. In one transgenic line (MOT1), the simian virus 40 large tumor antigen was expressed under the control of the mouse opsin promoter. MOT1 mice exhibited an age-related decline in the amplitude of the rod-mediated ERG a-wave. In comparison, cone-mediated responses recorded from MOT1 mice remained normal up to four months of age, the oldest age tested. In the second transgenic line (CMYC), the rat c-myc gene was expressed under control of the human interphotoreceptor-retinoid binding protein promoter. CMYC mice exhibited a rapid reduction of cone-mediated responses and a gradual loss of the rod ERG a-wave. Analysis of rod ERG a-waves obtained from young MOT1 and CMYC mice indicated that the rod ERG abnormalities reflect a reduction in the number of rods contributing to the response with the retention of normal response properties in rods that remain. These results support the possibility that aberrant expression of oncogenes may underlie some forms of human rod and cone-rod dystrophy.

Expression and Role of p53 in the Retina

Since it was identified in 1979, p53 has been widely studied for its role in tumor suppression. It is mutated in approximately half of all human cancers, leading to aberrant cell growth. In addition to its role as a tumor suppressor, p53 is activated in response to various cell stress signals, including DNA damage and hypoxia. This activation leads to alterations in target gene expression, giving p53 a regulatory role in diverse cellular functions such as apoptosis, senescence, and cell cycle arrest. Throughout life, the eye is exposed to a multitude of stressors including disease, light-induced damage, and oxidative stress, all of which can lead to debilitating loss of vision. This article examines the role of p53 during ocular development. Finally, the role of p53 is examined in ocular response to intense light exposure, ionizing radiation, oxidative stress, degenerative disorders, and retinoblastoma.