Differential distribution of interphotoreceptor retinoid-binding protein (IRBP) around retinal rod and cone photoreceptors

Light microscopic immunogold cytochemistry was used to examine the distribution of interphotoreceptor retinoid-binding protein (IRBP) in the interphotoreceptor matrix (IPM) surrounding rod and cone photoreceptors. Silver enhancement of retinas reacted with anti-IRBP antibodies using the two stage labeling procedure showed dense staining of the IPM around rod photoreceptor outer segments and the apical region of the RPE. However, the IPM around cone photoreceptors was lightly labeled for IRBP. This region of light labeling extended from the RPE to the distal one-fourth to one-third of the cone inner segment. Although most of the area surrounding cone outer segments was lightly labeled, a dense band of label was seen along the margins of cone outer segments. This study confirms that heterogeneity exists in the distribution of IRBP in the IPM and provides new evidence that IRBP is not homogeneously distributed around rod and cone photoreceptors.     

Flicker fusion characteristics of rod photoreceptors in the toad

Critical fusion frequency (CFF) of toad rods was examined at various intensities using intracellular recordings. Data were compared to CFF measurements (as a function of intensity) obtained with the electroretinogram (ERG) and with a horizontal cell. In all instances the rod (and rod mediated) responses produced a curve which increased monotonically to a high frequency asymptote at about 6 Hz. The curves obtained with the ERG and the horizontal cell were double branched. The upper branch exhibited a Purkinje shift and thus must have been produced by cones. Even when using a red background to suppress cone sensitivity, there was no sign of a second rod mediated branch in the CFF curves from this retina.     

Evolution of the genes mediating phototransduction in rod and cone photoreceptors

This paper reviews current knowledge of the evolution of the multiple genes encoding proteins that mediate the process of phototransduction in rod and cone photoreceptors of vertebrates. The approach primarily involves molecular phylogenetic analysis of phototransduction protein sequences, combined with analysis of the syntenic arrangement of the genes. At least 35 of these phototransduction genes appear to reside on no more than five paralogons – paralogous regions that each arose from a common ancestral region. Furthermore, it appears that such paralogs arose through quadruplication during the two rounds of genome duplication (2R WGD) that occurred in a chordate ancestor prior to the vertebrate radiation, probably around 600 millions years ago. For several components of the phototransduction cascade, it is shown that distinct isoforms already existed prior to WGD, with the likely implication that separate classes of scotopic and photopic photoreceptor cells had already evolved by that stage. The subsequent quadruplication of the entire genome then permitted the refinement of multiple distinct protein isoforms in rods and cones. A unified picture of the likely pattern and approximate timing of all the important gene duplications is synthesised, and the implications for our understanding of the evolution of rod and cone phototransduction are presented.     

A role for cytoskeletal elements in the light-driven translocation of proteins in rod photoreceptors

PURPOSE: Light-driven protein translocation is responsible for the dramatic redistribution of some proteins in vertebrate rod photoreceptors. In this study, the involvement of microtubules and microfilaments in the light-driven translocation of arrestin and transducin was investigated. METHODS: Pharmacologic reagents were applied to native and transgenic Xenopus tadpoles, to disrupt the microtubules (thiabendazole) and microfilaments (cytochalasin D and latrunculin B) of the rod photoreceptors. Quantitative confocal imaging was used to assess the impact of these treatments on arrestin and transducin translocation. A series of transgenic tadpoles expressing arrestin truncations were also created to identify portions of arrestin that enable arrestin to translocate. RESULTS: Application of cytochalasin D or latrunculin B to disrupt the microfilament organization selectively slowed only transducin movement from the inner to the outer segments. Perturbation of the microtubule cytoskeleton with thiabendazole slowed the translocation of both arrestin and transducin, but only in moving from the outer to the inner segments. Transgenic Xenopus expressing fusions of green fluorescent protein (GFP) with portions of arrestin implicates the C terminus of arrestin as an important portion of the molecule for promoting translocation. This C-terminal region can be used independently to promote translocation of GFP in response to light. CONCLUSIONS: The results show that disruption of the cytoskeletal network in rod photoreceptors has specific effects on the translocation of arrestin and transducin. These effects suggest that the light-driven translocation of visual proteins at least partially relies on an active motor-driven mechanism for complete movement of arrestin and transducin.     

Dark adaptation of rod photoreceptors in normal subjects, and in patients with Stargardt disease and an ABCA4 mutation

PURPOSE: Psychophysical and electroretinographic (ERG) studies indicate that patients with Stargardt disease exhibit abnormally slow rod dark adaptation after illumination that bleaches a substantial fraction of rhodopsin. However, relatively little information is available concerning rod recovery in this disease after weaker adapting (i.e., conditioning) light. With the use of a paired-flash ERG method, properties of the derived rod response to a low-bleach (<1%) but rod-saturating conditioning flash were investigated in seven normal subjects and in five Stargardt patients with identified sequence variations in the ABCA4 gene. METHODS: In the first of two experiments, the interval between a fixed conditioning flash (67 or 670 scotopic cd s m(-2)) and a bright probe flash of fixed strength was varied to determine the falling-phase kinetics of the derived rod response to the conditioning flash. In the second, the instantaneous amplitude-intensity function for the rod response at an intermediate stage of recovery from the conditioning flash was determined by presenting a test flash of various strengths at a fixed time after the conditioning flash, and a probe flash at 200 ms after the test flash. RESULTS: The maximum peak amplitude of the dark-adapted, rod-mediated a-wave determined in Stargardt patients (211 +/- 87 microV) was on average lower than that determined in normal subjects (325 +/- 91 microV; P = 0.06). The derived rod response to the 670 scotopic cd s m(-2) conditioning flash determined in normal subjects and Stargardt patients exhibited a biphasic recovery, and the kinetics of the early stage of this recovery were similar in the two subject groups. For both normal subjects and patients, normalized amplitude-intensity functions describing the dark-adapted derived rod response exhibited half-saturation at approximately 1.5 log scotopic troland second. In both groups, the normalized amplitude-intensity function determined at approximately 2 seconds after the 67 scotopic cd s m(-2) conditioning flash and at approximately 9 seconds after the 670 scotopic cd s m(-2) conditioning flash exhibited an average desensitization (i.e., an increase of test flash strength at half-saturation) of approximately 0.5 to 0.6 log unit relative to that determined under dark-adapted conditions. CONCLUSIONS: The results indicate that, despite a reduction in the average dark-adapted maximum a-wave amplitude in the Stargardt/ABCA4 patients, the early-stage recovery kinetics of the derived rod response to a low-bleaching conditioning flash as well as the lingering rod desensitization produced by such a flash are similar to those determined in normal subjects.     

A quantitative measure of the electrical activity of human rod photoreceptors using electroretinography

An electrical potential recorded from the cornea, the a-wave of the ERG, is evaluated as a measure of human photoreceptor activity by comparing its behavior to a model derived from in vitro recordings from rod photoreceptors. The leading edge of the ERG exhibits both the linear and nonlinear behavior predicted by this model. The capability for recording the electrical activity of human photoreceptors in vivo opens new avenues for assessing normal and abnormal receptor activity in humans. Furthermore, the quantitative model of the receptor response can be used to isolate the inner retinal contribution, Granit's PII, to the gross ERG. Based on this analysis, the practice of using the trough-to-peak amplitude of the b-wave as a proxy for the amplitude of the inner nuclear layer activity is evaluated.     

Regulation of calcium homeostasis in the outer segments of rod and cone photoreceptors

Calcium plays important roles in the function and survival of rod and cone photoreceptor cells. Rapid regulation of calcium in the outer segments of photoreceptors is required for the modulation of phototransduction that drives the termination of the flash response as well as light adaptation in rods and cones. On a slower time scale, maintaining proper calcium homeostasis is critical for the health and survival of photoreceptors. Decades of work have established that the level of calcium in the outer segments of rods and cones is regulated by a dynamic equilibrium between influx via the transduction cGMP-gated channels and extrusion via rod- and cone-specific Na⁺/Ca²⁺, K⁺ exchangers (NCKXs). It had been widely accepted that the only mechanism for extrusion of calcium from rod outer segments is via the rod-specific NCKX1, while extrusion from cone outer segments is driven exclusively by the cone-specific NCKX2. However, recent evidence from mice lacking NCKX1 and NCKX2 have challenged that notion and have revealed a more complex picture, including a NCKX-independent mechanism in rods and two separate NCKX-dependent mechanisms in cones. This review will focus on recent findings on the molecular mechanisms of extrusion of calcium from the outer segments of rod and cone photoreceptors, and the functional and structural changes in photoreceptors when normal extrusion is disrupted.     

Characterization of the interphotoreceptor matrix surrounding rod photoreceptors in the human retina.

Previous studies have documented the presence of specific lectin-binding domains in the insoluble interphotoreceptor matrix (IPM) isolated from human retina. Peanut agglutinin (PNA) selectively binds to cone matrix domains whereas wheat germ agglutinin (WGA) binds to matrix domains surrounding rods. In the present study, the rod-associated WGA-binding domains are further characterized using lectin-based cytochemistry and polyacrylamide gel electrophoresis in combination with neuraminidase digestion. The lectin-binding patterns of non-neuraminidase-treated samples are similar to those described in previous reports. After neuraminidase treatment, both rod and cone matrix domains demonstrate PNA binding while the binding of WGA to rod matrix domains is reduced. However, the binding of WGA to photoreceptor outer segments is not affected by neuraminidase. Blots of IPM proteins probed with lectins indicate that the WGA-binding macromolecules are represented as a group of high molecular weight glycoproteins, whereas the PNA-binding components are represented as a group of lower molecular weight glycoproteins. The major WGA-binding glycoprotein (147 kDa) shows reduced binding affinity to WGA and increased binding affinity to PNA following neuraminidase treatment. Further, this 147-kDa glycoprotein, although similar in molecular weight to IRBP (interphotoreceptor retinol-binding protein) (141 kDa), is not recognized by the lectin, concanavalin A (Con A), or by an anti-IRBP antibody. Our data suggest that: (1) the major component of the WGA-binding domain demonstrated by polyacrylamide gel analysis is a glycoprotein with a molecular weight of 147 kDa containing galactose residues that are masked by terminal sialic acid residues; and (2) the binding of WGA to photoreceptor outer segments is resistant to neuraminidase, consistent with the earlier reports that WGA-binding domains of photoreceptor outer segments may not be sialyl-containing glycoconjugates.     

GDNF stimulates rod photoreceptors and dopaminergic amacrine cells in chicken retinal reaggregates

PURPOSE: To investigate the role(s) of glial cell line-derived neurotrophic factor (GDNF) on expression of rod photoreceptor and dopaminergic amacrine cell-specific genes in an in vitro reaggregate model of the chick retina. METHODS: Retinal reaggregates derived from embryonic day (E)6 chicks (rosetted spheroids) were supplemented with 50 ng/mL GDNF, or, alternatively, endogenous GDNF expression was downregulated by transient transfection of spheroids with a pCMS-EGFP[GDNF] antisense vector. Using mainly semiquantitative RT-PCR analyses, expression of rhodopsin, four separate opsins, and tyrosine hydroxylase (THase) was analyzed after either treatment. RESULTS: Supplementation with GDNF accelerated rhodopsin mRNA expression and sustained it at an increased level, in contrast to untreated control subjects, where rhodopsin mRNA levels were lower and unmaintained. Expression of red, green, blue, and violet opsins were unaffected. Under these conditions, GDNF also massively increased the expression of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of dopamine. The expression of endogenous GDNF was blocked in spheroids by using antisense transfections, which resulted in both a significant decrease in rhodopsin mRNA expression and a complete suppression of THase expression, as determined by RT-PCR, Western blot analysis, and immunocytochemistry. CONCLUSIONS: GDNF supports expression of both rhodopsin and THase in vitro, two critical molecules involved in the production of rod photoreceptors and dopaminergic amacrine cells, respectively; however, the presence of GDNF does not affect cone production and survival.     

Effects of light and darkness on pH outside rod photoreceptors in the cat retina.

We recorded pH in the extracellular space surrounding rod photoreceptors in the dark-adapted eye of the cat and during illumination with double-barreled H(+)-selective microelectrodes. A pH of 7.17 was recorded in the vitreous at the retinal surface of the dark-adapted eye and this became more alkaline during light adaptation. In dark adaptation, a pH close to 7.00 was recorded in a region of maximal acidity in the extracellular space surrounding rods in the outer nuclear layer (ONL). pH steeply alkalinized as the microelectrode was moved more distally towards the retinal pigment epithelium (RPE), and almost reached the pH of the arterial blood at the apical surface of the RPE. Illumination produced an intraretinal alkalinization that was largest (up to 0.2 pH units) in the ONL, maximal in amplitude at rod-saturating intensities, and that was sustained during steady background illumination. The light-evoked alkalinization was relatively slow in onset, having a time constant (1/e) of 64 sec, and took 8-12.5 min to return to the dark-adapted level after the offset of maintained illumination. These results show that acid production by cat rods is highest in the dark, reflecting a high rate of energy metabolism, and suggest that glycolysis is required to support the dark current. Illumination, by suppressing both glycolysis and respiration, alkalinizes the extracellular space surrounding rods. The substantial change in pH outside rods from dark to light could alter pH dependent properties of the interphotoreceptor matrix.     

Effects of intraocular pressure on pH outside rod photoreceptors in the cat retina.

Double-barreled H(+)-selective microelectrodes were used to study the effect of raising intraocular pressure (ocular hypertension) on intraretinal pH in the cat. A surprising alkalinizing response was observed in the subretinal space on the first experimental day in seven of 11 experiments. Of the three retinal regions studied, the alkalinizing response was present in the area centralis and in the adjoining near-discal region, but was absent in the superior temporal periphery. The alkalinizing response was relatively fragile, diminishing and then extinguishing with repetition of episodes of ocular hypertension. It was also never observed on the second experimental day. The alkalinizing response had a relatively low threshold, appearing with 10 mmHg elevations of intraocular pressure, i.e. when the perfusion pressure was still relatively high, but was also present across a wide range of perfusion pressures. It was maximal in amplitude in the most distal portion of the subretinal space and consisted of two components, an 'on' alkalinization during the episode of ocular hypertension, and an 'off' alkalinization following it. When the alkalinizing response was present in the subretinal space, it also could be recorded with placement of the microelectrode in the choroid. Elevations of systemic arterial blood pressure with epinephrine (intravenous), by raising perfusion pressure, also alkalinized the subretinal space, indicating that an increase in choroidal blood flow can produce this type of pH response. While pH recordings do not directly monitor blood flow, we tentatively interpreted the alkalinizing response with ocular hypertension as originating from a regional increase in choroidal blood flow, but we cannot rule out other explanations. Acidification of the subretinal space in response to ocular hypertension replaced the alkalinizing response when the latter extinguished, or acidification was the sole response in the absence of an alkalinization. The acidification also had a low threshold, was maximal in the most distal portion of the subretinal space, and increased in size with decreases in perfusion pressure. The acidification was reduced in amplitude by breathing 100% oxygen (hyperoxia). This was interpreted as suppression of the component of acidification that originated from an increase in glycolysis by the rod photoreceptors during ocular hypertension.     

Cloning and molecular characterization of cGMP-gated ion channels from rod and cone photoreceptors of striped bass ( M. saxatilis ) retina

Vertebrate photoreceptors respond to light with changes in membrane conductance that reflect the activity of cyclic-nucleotide gated channels (CNG channels). The functional features of these channels differ in rods and cones; to understand the basis of these differences we cloned CNG channels from the retina of striped bass, a fish from which photoreceptors can be isolated and studied electrophysiologically. Through a combination of experimental approaches, we recovered and sequenced three full-length cDNA clones. We made unambiguous assignments of the cellular origin of the clones through single photoreceptor RT-PCR. Synthetic peptides derived from the sequence were used to generate monospecific antibodies which labeled intact, unfixed photoreceptors and confirmed the cellular assignment of the various clones. In rods, we identified the channel alpha subunit gene product as 2040 bp in length, transcribed into two mRNA 1.8 kb and 2.9 kb in length and translated into a single 96-kDa protein. In cones we identified both alpha (CNGA3) and beta (CNGB3) channel subunits. For alpha, the gene product is 1956 bp long, the mRNA 3.4 kb, and the protein 74 kDa. For beta, the gene product is 2265 bp long and the mRNA 3.3 kb. Based on deduced amino acid sequence, we developed a phylogenetic map of the evolution of vertebrate rod and cone CNG channels. Sequence comparison revealed channels in striped bass, unlike those in mammals, are likely not N-linked-glycosylated as they are transported within the photoreceptor. Also bass cone channels lack certain residues that, in mammals, can be phosphorylated and, thus, affect the cGMP sensitivity of gating. On the other hand, functionally critical residues, such as positively charged amino acids within the fourth transmembrane helix (S4) and the Ca(2+)-binding glutamate in the pore loop are absolutely the same in mammalian and nonmammalian species.     

Immunolocalization of 48K in rod photoreceptors. Light and ATP increase OS labeling

An abundant, light-modified protein of Mr approximately equal to 51 kD, homologous to a previously described protein (termed 48K, arrestin, or S-antigen) of bovine retina, was identified and characterized in rod photoreceptors of the toad (Bufo marinus). Isolated, intact retinas were incubated in darkness, or irradiated under physiological conditions [dark-adapted (DA) and light-adapted (LA) retinas]. Using polyclonal antibodies raised in rabbit against purified toad 48K, and post-embedding immunoelectron microscopy (second antibody conjugated with 5 nm gold particles), we examined the localization of 48K in DA vs. LA rods. Physiological incubations and early fixation steps were carried out in the presence vs. absence of ATP (40 microM), a substance thought to support the activity of 48K in vivo. The distribution of 48K in rods was analyzed by quantitating the density of gold label within subcellular compartments. In DA rods, labeling was highest in the myoid region of the inner segment. Light adaptation increased 48K immunoreactivity within the outer segment, and decreased labeling within calycal processes. Labeling in outer segments of LA rods was maximal in the basal region. Treatment with ATP both amplified the light-dependent increase in basal OS labeling, and augmented the nonuniformity of basal vs. apical labeling in LA ROS. Morphometric analysis of labeling density over the length of DA vs. LA ROS indicated an approximately equal to 1.5-fold net increase in 48K label in LA ROS. By comparison, biochemical analysis of 48K level indicated an approximately equal to 1.8-fold increase in 48K in LA preparations. Together, the biochemical and immunocytochemical data indicate that the relative amount of 48K is increased in LA ROS, not just its immunoreactivity. The data are consistent with a selective movement of 48K, a cytoplasmic ROS protein, into ROS during LA.     

Early apoptosis of rod photoreceptors in Rpe65(-/-) mice is associated with the upregulated expression of lysosomal-mediated autophagic genes

RPE65-related Leber's congenital amaurosis (LCA) is a rod-cone dystrophy whose clinical outcome is mainly attributed to the loss of rod photoreceptors followed by cone degeneration. Pathogenesis in Rpe65(-/-) mice is characterized by a slow and progressive degeneration of rods dependent on the constitutive activation of unliganded opsin. We previously reported that this opsin-mediated apoptosis of rods was dependent on Bcl-2-apoptotic pathway and Bax-induced pro-death activity. In this study, we report early initial apoptosis in the newly differentiated retina of Rpe65(-/-) mice. Apoptotic photoreceptors were identified as rods and resulted from pathological phototransduction signaling. This wave of early apoptosis triggered Bcl-2-related pathway and Bax apoptotic activity, while activation of the caspases was not induced. Following cellular stress, multiple signaling pathways are initiated which either commit cells to death or trigger pro-survival responses including autophagy. We report that Bcl-2-related early rod apoptosis was associated with the upregulation of autophagy markers including chaperone-mediated autophagy (CMA) substrate receptor LAMP-2 and lysosomal hydrolases Cathepsin S and Lysozyme. This suggests that lysosomal-mediated autophagy may be triggered in response to early rod apoptosis in Rpe65-LCA disease. These results highlight that Rpe65-related primary stress induces early signaling events, which trigger Bax-induced-apoptotic pathway and autophagy-mediated cellular response. These events may determine retinal cell fate, progression and severity of the disease.     

A nonsense mutation in the glucosaminyl (N-acetyl) transferase 2 gene (GCNT2): association with autosomal recessive congenital cataracts

PURPOSE: To identify the genetic defect associated with autosomal recessive congenital cataract in four Arab families from Israel. METHODS: Genotyping was performed using microsatellite markers spaced at approximately 10 cM intervals. Two-point lod scores were calculated using MLINK of the LINKAGE program package. Mutation analysis of the glucosaminyl (N-acetyl) transferase 2 gene (GCNT2) gene was performed by direct sequencing of PCR-amplified exons. RESULTS: The cataract locus was mapped to a 13.0-cM interval between D6S470 and D6S289 on Chr. 6p24. A maximum two-point lod score of 8.75 at theta = 0.019 was obtained with marker D6S470. Sequencing exons of the GCNT2 gene, mutations of which have been associated with cataracts and the i blood group phenotype, revealed in these families a homozygous G-->A substitution in base 58 of exon-2, resulting in the formation of premature stop codons W328X, W326X, and W328X, of the GCNT2A, -B, and -C isoforms, respectively. Subsequent blood typing of affected family members confirmed the possession of the rare adult i blood group phenotype. CONCLUSIONS: A nonsense mutation in the GCNT2 gene isoforms is associated with autosomal recessive congenital cataract in four distantly related Arab families from Israel. These findings provide further insight into the dual role of the I-branching GCNT2 gene in the lens and in reticulocytes.