Cyclic guanosine monophosphate: elevation in degenerating photoreceptor cells of the C3H mouse retina.

As a result of an early deficiency in cyclic nucleotide phosphodiesterase activity, guanosine 3',5'-monophosphate accumulates in retinal photoreceptor cells before they begin to degenerate. It is suggested that degeneration of the photoreceptor cells is related to an imbalance in their metabolism or function which is caused by the elevated levels of cyclic guanosine monophosphate.

Cyclic GMP accumulation causes degeneration of photoreceptor cells: simulation of an inherited disease.

Guanosine 3',5'-monophosphate (cyclic GMP) metabolism in developing eye rudiments of Xenopus laevis embryos in culture is disrupted by the phosphodiesterase inhibitor isobutylmethylxanthine. At low concentrations of inhibitor the rudiments develop normally, but at higher concentrations of the inhibitor, cyclic GMP accumulates in the rudiments and the retinal photoreceptor cells degenerate selectively. The isobutylmethylxanthine-induced photoreceptor degeneration is associated with an accumulation of cyclic GMP and, in this respect, it stimulates an early biochemical defect in the inherited degenerative disease of rd mice.

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.

The beta subunit of cyclic GMP phosphodiesterase mRNA is deficient in canine rod-cone dysplasia 1.

Irish setter dogs affected with rod-cone dysplasia 1 have elevated levels of retinal cGMP resulting from deficient rod-specific cGMP phosphodiesterase (cGMP PDE) activity. We investigated the mRNAs coding for the three subunits of cGMP PDE and for the proteins involved in the activation/deactivation of this enzyme in the retinas of developing affected and control dogs. While the photoreceptor cells are viable in the diseased retinas, opsin, transducin alpha 1 and beta 1, 48 and 33 kd proteins, and cGMP PDE alpha and gamma mRNAs have normal transcript sizes and levels. In contrast, a different pattern of cGMP PDE beta mRNAs with lower than normal concentrations is present in the developing affected retinas prior to degeneration. Our observations suggest that an abnormality involving cGMP PDE beta expression is implicated in rod-cone dysplasia 1.

Identification of genes causing photoreceptor degenerations leading to blindness.

At least 15 genes with defects responsible for various forms of inherited retinal disease involving photoreceptor loss have been identified over the past eight years. Several of the genes were first considered as candidates for study because of their involvement in murine retinal disease, others because of their chromosomal loci. In two cases, novel genes were uncovered by positional cloning. Based on reports of disease loci for which no gene has yet been found, more than twice as many genes remain to be identified in this genetically heterogeneous group of diseases.

Isolation and characterization of a cDNA encoding the alpha' subunit of human cone cGMP-phosphodiesterase.

A human cDNA (alpha-PDE) encoding the alpha' catalytic subunit of cone photoreceptor cGMP-phosphodiesterase has been isolated and characterized. The nucleotide sequence of 2980 bp contains an open reading frame encoding an 859-amino-acid (aa) protein with a calculated molecular mass of 99 kDa. Northern blot analysis of human retinal mRNA hybridized with the alpha'-PDE cDNA revealed a signal corresponding to a 3.2-kb transcript. Comparison of the deduced aa sequence of human cone alpha'-PDE with corresponding proteins isolated from bovine and chicken retinas shows 89 and 83% identity, respectively, and indicates that alpha'-PDE has been very well conserved in the evolutionary process. Human cone alpha'-PDE is highly homologous to rod alpha-PDE and beta-PDE, suggesting that these proteins may have a close phylogenetic relationship.

Transcription factor IID probes localize a single gene to the proximal region of mouse chromosome 17.

We have used a 5' fragment of the gene GTF2D, which encodes human transcription factor IID, and Chinese hamster-mouse somatic cell hybrids to map the murine homologue, Gtf2d, to a single locus on mouse chromosome 17 (Chr 17). Linkage analysis of progeny from an interspecific backcross localized the gene near the marker D17Leh66 in the proximal region of Chr 17.

Isolation and characterization of cDNA encoding the gamma-subunit of cGMP phosphodiesterase in human retina.

Cyclic GMP-phosphodiesterase (cGMP-PDE) plays a key role in the normal functioning of retinal rod photoreceptor cells. The enzyme is composed of alpha- and beta-catalytic subunits which are inhibited by two identical gamma-subunits. A cDNA encoding the gamma-subunits (PDE gamma) from human retina has been cloned and sequenced. The 1012-bp cDNA has a coding region of 261 bp which is highly homologous to those of the PDE gamma cDNAs from bovine and mouse retinas. Comparison of the deduced amino acid sequences of the proteins from the three species indicates that PDE gamma has been very well conserved through evolution. The mRNA encoded by the cloned cDNA is 1.0 kb long, is similar in size to the corresponding mRNAs from mouse, dog and bovine retinas and is not detected in ground squirrel retina. The PDEG gene has been assigned to human chromosome 17, probably in the region q21.1.

Structure and analysis of the transducin beta3-subunit gene, a candidate for inherited cone degeneration (cd) in the dog.

The cDNA for the beta3-subunit of cone-specific transducin (Tbeta3) was cloned and characterized from wild type dogs, and used in linkage studies as a candidate gene for cone degeneration. Sequence analysis of the Tbeta3 cDNA revealed an open reading frame of 1020 bp, potentially coding for a protein of 340 amino acids (aa). The deduced aa sequence of canine Tbeta3 shares 97% identity with the previously identified human Tbeta3, and 82% identity with bovine rod-specific transducin (Tbeta1). RT-PCR and sequencing of the amplified products demonstrated that the retinal canine Tbeta3 gene is expressed in two different transcripts which can be generated by alternative splicing of the intron in the 3'-untranslated region (UTR). The short and the long mRNAs differ in the length of their 3'-UTR by 456 nt. We have also determined the genomic organization of the canine Tbeta3 gene; it consists of ten exons and the first exon is in the 5'-UTR. The cDNA encoding Tbeta3 from cd-affected dogs was also cloned and sequenced. We found no differences at the nucleotide level between the cDNAs isolated from normal and diseased retinas. The level of transcription of Tbeta3 mRNA in the cd dog retina appeared to be normal. Linkage analysis of a crossbred informative pedigree showed five obligate recombinants out of nine informative offspring. These results suggest that Tbeta3 is not a candidate gene for the cone degeneration of the cd mutant.

The mouse X-linked juvenile retinoschisis cDNA: expression in photoreceptors.

Retinal photoreceptor cells are particularly vulnerable to degenerations that can eventually lead to blindness. Our purpose is to identify and characterize genes expressed specifically in photoreceptors in order to increase our understanding of the biochemistry and function of these cells, and then to use these genes as candidates for the sites of mutations responsible for degenerative retinal diseases. We have characterized a cDNA, a fragment of which (SR3.1) was originally isolated by subtractive hybridization of adult, photoreceptorless rd mouse retinal cDNAs from the cDNAs of normal mouse retina. The full-length sequence of this cDNA was determined from clones obtained by screening mouse retinal and eye cDNA libraries and by using the 5'- and 3'-RACE methods. Both Northern blot analysis and in situ hybridization showed that the corresponding mRNA is expressed in rod and cone photoreceptors. The gene encoding this cDNA was mapped to the X chromosome using an interspecific cross. Based on the nucleotide and amino acid sequences, as well as chromosome mapping, we determined that this gene is the mouse ortholog (Xlrs1) of the human X-linked juvenile retinoschisis gene (XLRS1). Analysis of the predicted amino acid sequence indicates that the Xlrs1 mRNA may encode a secretable, adhesion protein. Therefore, our data suggest that X-linked juvenile retinoschisis originates from abnormalities in a photoreceptor-derived adhesion protein.

Gamma-subunit of mouse retinal cyclic-GMP phosphodiesterase: cDNA and corresponding amino acid sequence.

The cDNA nucleotide and corresponding amino acid sequences of the gamma-subunit of cyclic-GMP phosphodiesterase (cGMP-PDE gamma) from mouse retina have been determined. The cDNA translated region was found to be 91.5% homologous to the cDNA coding region for the enzyme from bovine retina [(1986) FEBS Lett. 204, 288-292]. On Northern blots of normal mouse retinal RNAs this cDNA hybridized the cGMP-PDE gamma mRNA which is 900 bp long. The mouse gamma-subunit contains 87 amino acid residues which share 97.7% homology with the bovine polypeptide [(1986) FEBS Lett. 204, 288-292]. Only two amino acids have been changed, Ala 8 to Gly and Met 17 to Ile.

Autophosphorylation of calmodulin kinase II: functional aspects.

Autophosphorylation of purified calmodulin kinase II dramatically inhibited protein kinase activity and enhanced substrate selectivity. Inhibition was observed over a wide range of calmodulin concentrations but calmodulin binding was unaffected. Autophosphorylation of calmodulin kinase II may be a mechanism for limiting phosphorylation to physiological substrates and terminating some of calcium's actions in synaptic events.

Conserved transcriptional regulation of a cone phototransduction gene in vertebrates.

cGMP-phosphodiesterase (PDE) is a key component in visual phototransduction. Rod and cone photoreceptors each produce their unique cGMP-PDE subunits. The alpha' catalytic subunits are believed to be cone-specific. In this study, we report that transfection of the -132 to +139 sequence in the upstream region of the human alpha'-PDE gene fused to luciferase cDNA gives the highest level of reporter gene transcription in cultured retinoblastoma Y79 cells. Transgenic Xenopus laevis carrying this sequence fused to green fluorescent protein (GFP) expressed GFP in cones, suggesting a conserved regulatory mechanism for alpha'-PDE transcription in both human and frog.

Isolation and characterization of a novel oligodendrocyte-specific protein.

Myelin plays a critical role in nervous system function and alterations in myelin-specific proteins cause a variety of neurologic disorders. We isolated a novel cDNA from the CNS that shares little nucleotide sequence homology with previously reported genes but appears to encode a protein related to peripheral myelin protein-22 (PMP-22) based on its amino acid sequence, predicted structure, and cellular localization. PMP-22 is important in peripheral myelination and Schwann cell proliferation, and mutations in its gene cause diseases of peripheral nerves. The isolated cDNA is 1.8 kb in length with an open reading frame of 621 bp. Northern blot analysis detected hybridization of labeled cDNA with a single 2.1-kb transcript only in the CNS. In situ hybridization revealed expression of this cDNA in oligodendrocytes of brain and spinal cord as well as in oligodendrocyte-enriched cultures; therefore we have named it oligodendrocyte-specific protein (OSP) cDNA. An OSP-specific polyclonal antibody reacted with a single 22-kd protein present in CNS myelin and oligodendrocytes. Developmental expression of OSP mRNA in the spinal cord was similar to that of the mRNA for a major myelin protein, proteolipid protein (PLP), and similar to PMP-22 in peripheral nerves. Since OSP is localized to oligodendrocytes and myelin, has a similar structure with PMP-22, and has a developmental pattern of expression like other myelin proteins, it probably has an important role in CNS myelinogenesis.

Rhodopsin phosphorylation in developing normal and degenerative mouse retinas.

The developmental pattern of rhodopsin phosphorylation in degenerative (rdle homozygote) and normal (rd/+ heterozygote) mouse retina has been investigated. The results indicate that rhodopsin levels are comparable in the 2 retinas up to about 10 days of age but that rhodopsin phosphorylation is not. The phosphorylation of rhodopsin is substantially reduced in the degenerative retina during development. This abnormality may be an expression of the rd lesion. The rhodopsin kinase/phosphatase system, the G protein, and the visual pigment are all involved in the modulation of cGMP-phosphodiesterase activity in normal retinas. A defect in any of these components could account for the reduced level of cGMP-phosphodiesterase activity in rd retinas, resulting in cGMP accumulation and subsequent photoreceptor degeneration.

Photoreceptor degeneration in a pure-cone retina. Effects of cyclic nucleotides, and inhibitors of phosphodiesterase and protein synthesis.

Previous studies have shown that disruption of cyclic nucleotide metabolism by phosphodiesterase inhibitors and cyclic nucleotide analogues damages photoreceptors in rod-enriched retinae. In these studies the cone photoreceptors appeared damaged only after the surrounding rods had begun to degenerate. Our aim was to test if cone photoreceptors were susceptible to similar treatments in the absence of rod photoreceptors. We treated pure-cone lizard retinae in an in vitro eyecup preparation. Degeneration of the cones was induced by 10(-3) M, but not 10(-5) M, of the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX). Changes in the morphology of the cone outer segments were first evident after 10 hr at 24 degrees C. After longer exposures, other retinal cells were also affected. Before morphology was affected, synthesis of proteins of all molecular weights was inhibited throughout the retina. In addition, both retinal cyclic AMP and cyclic GMP levels were elevated, particularly after 2-10 hr. The effects of 10(-3) M IBMX on all of these parameters were still reversible by removal from IBMX after 10 hr. Dibutyryl cyclic AMP at 10(-2) M also inhibited protein synthesis. It also induced degeneration, but less rapidly than 10(-3) M IBMX. Dibutyryl cyclic GMP (10(-2) M) or butyric acid did not significantly affect morphology, or inhibit uptake or incorporation of 3H-leucine by retinae. The concentration of puromycin or cycloheximide that inhibited retinal protein synthesis by the same amount as 10(-3) M IBMX did not affect retinal morphology or cyclic nucleotide levels. One hundred times this concentration induced pyknosis in the nuclear layers of the retina before disturbing cone outer segment organization. In conclusion, millimolar IBMX and dibutyryl cyclic AMP damage cones even without neighboring rods, indicating that elevated cyclic nucleotide levels are toxic to cones per se. Retinal protein synthesis is also inhibited by damaging levels of cyclic nucleotides, but it does not seem to be responsible for the deterioration of cone structure.

Beta adrenergic receptors on cultured human retinal pigment epithelium.

Cultured fetal human retinal pigment epithelium (RPE) was grown on a permeable substrate and sealed in an Ussing chamber. The average electrical resistance (R) was 330 ohm-cm2, the average transepithelial voltage (Ve) was 3.0 mV (apical side positive), and the average short circuit current (Isc) was 9.1 microA/cm2. When these RPE preparations were exposed to isoproterenol (a beta-adrenergic agonist), the Isc increased by 88%, R was reduced by 6%, and Ve increased by 85%. The effect of isoproterenol was blocked by propranolol (a beta-adrenergic antagonist). When cultured human RPE was exposed to isoproterenol, intracellular cyclic adenosine monophosphate (AMP) levels rose more than threefold. The effect of isoproterenol on cyclic AMP levels was blocked by propranolol. When the cultured RPE was exposed to dibutyryl cyclic AMP, both Ve and Isc rose by 47% with a time course similar to that which occurred when the cells were exposed to isoproterenol. Preparations treated with dibutyryl cyclic AMP did not respond to subsequently applied isoproterenol. These results indicate that cultured human RPE possesses a beta-adrenergic receptor and that stimulation of this receptor produces a change in cyclic AMP concentration which affects RPE electrical activity.

Distribution patterns of photoreceptors, protein, and cyclic nucleotides in the human retina.

The concentration of cGMP, cAMP, protein and the number of cone and rod photoreceptors have been measured in parallel arrays of punches, 3 mm in diameter, taken from each quadrant of normal human retinas. A separate punch containing the fovea and parafoveal region was also analyzed. Eyes were obtained from four male donors ranging in age from 35 to 67 yr. The retina thins considerably from the center to the periphery, and consequently the protein content forms a gradient in the same direction. Similar gradients were observed for cAMP and cGMP concentrations. In all eyes studied, the foveal-parafoveal region had higher levels of cAMP than cGMP. The data was analyzed with the aid of a computer in order to obtain three-dimensional maps of the patterns of distribution of the different parameters. A strong correlation between the areas of higher cone density, non-photoreceptor neurons, and cAMP, and an equally strong correlation between rod distribution and that of cGMP was observed. These maps will serve as baseline data in studies of pathological conditions such as retinitis pigmentosa.

Cone visual cell degeneration in ground squirrel retina: disruption of morphology and cyclic nucleotide metabolism by lodoacetic acid.

Photoreceptor degeneration was induced in the cone-dominant retina of the ground squirrel by intracardiac injection of iodoacetic acid. Morphology was examined and cyclic nucleotide levels were determined in retinas taken at various times between 35 min and 11 days after injection. Degenerating cone cells were first detected at day 1 and all cone cells were reduced to compact dense masses by day 4. Cellular debris was removed by macrophages that entered the photoreceptor layer from the inner neuroretina. Cyclic AMP levels of dark-adapted retinas were doubled 24 hrs after injection and were reduced to approximately 50% of the dark-adapted level of control retina between days 1 and 3. The concentration of cyclic GMP was 3 to 4 times higher than normal at 4 to 5 hrs postinjection, dropped to 9% of normal at day 4, and was 2% at day 11. Since these changes were coincident with the loss of morphologic integrity of cone cells, they imply that at least 50% of cyclic AMP and most of the cyclic GMP in the cone-dominant retina of the ground squirrel is present in cone cells. The elevation of cyclic AMP and cyclic GMP levels prior to pathological morphology suggests that the iodoacetic acid-induced disruption of cyclic nucleotide metabolism may be associated with the degeneration of the cone photoreceptors.

Light-induced phosphorylation of proteins from the all-cone retina of the lizard, Anolis carolinensis.

Proteins from the all-cone retina of the lizard Anolis carolinensis were phosphorylated using [gamma 32P] ATP, separated by SDS-PAGE and detected by autoradiography. Several proteins incorporated 32P. Exposure of the retinal homogenates to light brought about a dramatic increase in phosphorylation of the protein(s) with a molecular weight nearly identical to that of rat rhodopsin. It is likely that these proteins are the cone visual pigments, and that they incorporate phosphate when bleached by light. Increasing the time of the phosphorylation reaction from 1 to 30 min led to an increase in the amount of incorporation of labeled phosphate by the putative cone visual pigments, but changing the temperature from 4 degrees C to 20 degrees C decreased it. The amount of phosphate incorporation was substantially increased by NaF, a phosphatase inhibitor. This latter finding, along with the changes in incorporation of 32P with increased temperature, suggest that a phosphoprotein phosphatase is active in the lizard retina. The cation requirements, as well as the effects of cyclic nucleotides on light-induced phosphorylation of retinal lizard proteins, were also investigated.