Disease sequence from mutant rhodopsin allele to rod and cone photoreceptor degeneration in man

Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, lead to retinal degeneration in species from Drosophila to man. The pathogenic sequence from rod cell-specific mutation to degeneration of rods and cones remains unclear. To understand the disease process in man, we studied heterozygotes with 18 different rhodopsin gene mutations by using noninvasive tests of rod and cone function and retinal histopathology. Two classes of disease expression were found, and there was allele-specificity. Class A mutants lead to severely abnormal rod function across the retina early in life; topography of residual cone function parallels cone cell density. Class B mutants are compatible with normal rods in adult life in some retinal regions or throughout the retina, and there is a slow stereotypical disease sequence. Disease manifests as a loss of rod photoreceptor outer segments, not singly but in microscopic patches that coalesce into larger irregular areas of degeneration. Cone outer segment function remains normal until >75% of rod outer segments are lost. The topography of cone loss coincides with that of rod loss. Most class B mutants show an inferior-nasal to superior-temporal retinal gradient of disease vulnerability associated with visual cycle abnormalities. Class A mutant alleles behave as if cytotoxic; class B mutants can be relatively innocuous and epigenetic factors may play a major role in the retinal degeneration.     

Intestinal bleeding associated with systemic amyloidosis

Total colourblindness (OMIM 216900), also referred to as rod monochromacy (RM) or complete achromatopsia, is a rare, autosomal recessive inherited and congenital disorder characterized by photophobia, reduced visual acuity, nystagmus and the complete inability to discriminate between colours. Electroretinographic recordings show that in RM, rod photoreceptor function is normal, whereas cone photoreceptor responses are absent. The locus for RM has been mapped to chromosome 2q11 (ref. 2), however the gene underlying RM has not yet been identified. Recently, a suitable candidate gene, CNGA3, encoding the alpha-subunit of the cone photoreceptor cGMP-gated cation channel, a key component of the phototransduction pathway, has been cloned and assigned to human chromosome 2q11 (refs 3,4). We report the identification of missense mutations in CNGA3 in five families with RM. Homozygous mutations are present in two families, whereas the remaining families show compound heterozygous mutations. In all cases, the segregation pattern of the mutations is consistent with the autosomal recessive inheritance of the disease and all mutations affect amino acids that are highly conserved among cyclic nucleotide gated channels (CNG) in various species. This is the first report of a colour vision disorder caused by defects other than mutations in the cone pigment genes, and implies at least in this instance a common genetic basis for phototransduction in the three different cone photoreceptors of the human retina.     

Pathogenesis of neuroimmunologic diseases. Experimental models

Development of the retina, like that of other tissues, occurs via an orderly sequence of cell division and differentiation, producing the functional retina. In teleost fish, however, cell division and differentiation in the retina continue throughout the life of the animal in two distinct ways. Stem cells in a circumferential germinal zone at the periphery of the retina give rise to all retinal cell types and progenitor cells located throughout the retina in the outer nuclear layer (ONL) produce new rod photoreceptors. These processes in adult retina recapitulate in space the embryonic events responsible for forming the retina. Analysis of these events in an African cichlid fish, Haplochromis burtoni, confirmed that cone photoreceptors differentiate first, followed by rod photoreceptors. Correspondingly, at the margin of the eye, cone photoreceptors differentiate nearer to the margin than do rods. Control of photoreceptor production is not understood. Here we present the time of appearance and distribution pattern of GABA and vimentin which are candidates for the control of retinal cell division and differentiation. Antibody staining reveals that both GABA and vimentin exhibit unique patterns of expression during embryonic retinal development. Vimentin immunoreactivity is evident throughout the retina in a spoke-like pattern between developmental Days 4 and 7, as both cone and rod photoreceptors are being formed. GABA is expressed in horizontal cells between Days 5 and 7, corresponding to the onset of rod differentiation in time and in position within the retina. Moreover, the wave of GABAergic staining in the horizontal cells parallels the wave of rod differentiation across the embryonic retina of H. burtoni. Thus, GABA may play a role in the development of rod photoreceptors.     

Immunocytochemical localization of the NMDA-R2A receptor subunit in the cat retina

Immunocytochemical studies were performed to determine the distribution and cellular localization of the NMDA-R2A receptor subunit (R2A) in the cat retina. R2A-immunoreactivity (R2A-IR) was noted in all layers of the retina, with specific localizations in the outer segments of red/green and blue cone photoreceptors, B-type horizontal cells, several types of amacrine cells, Müller cells and the majority of cells in the ganglion cell layer. In the inner nuclear layer, 48% of all cells residing in the amacrine cell layer were R2A-IR including a cell resembling the GABAergic A17 amacrine cell. Interestingly, the AII rod amacrine cell was devoid of R2A-IR. Although the localization of the R2A subunit was anticipated in ganglion cells, amacrines and Müller cells, the presence of this receptor subunit to the cells in the outer retina was not expected. Here, both the R2A and the R2B subunits were found to be present in the outer segments of cone photoreceptors and to the tips of rod outer segments. Although the function of these receptor subunits in rod and cone photoreceptors remains to be determined, the fact that both R2A and R2B receptor subunits are localized to cone outer segments suggests a possible alternative pathway for calcium entry into a region where this cation plays such a crucial role in the process of phototransduction. To further classify the cells that display NR2A-IR, we performed dual labeling experiments showing the relationship between R2A-labeled cells with GABA. Results showed that all GABAergic-amacrines and displaced amacrines express the R2A-subunit protein. In addition, approximately 11% of the NR2A-labeled amacrines, did not stain for GABA. These findings support pharmacological data showing that NMDA directly facilitates GABA release in retina and retinal cultures [I.L. Ferreira, C.B. Duarte, P.F. Santos, C.M. Carvalho, A.P. Carvalho, Release of [3H]GABA evoked by glutamate receptor agonist in cultured chick retinal cells: effect of Ca2+, Brain Res. 664 (1994) 252-256; G.D. Zeevalk, W.J. Nicklas, Action of the anti-ischemic agent ifenprodil on N-methyl-d-aspartate and kainate-mediated excitotoxicity, Brain Res. 522 (1990) 135-139; R. Huba, H.D. Hofmann, Transmitter-gated currents of GABAergic amacrine-like cells in chick retinal cultures, Vis. Neurosci. 6 (1991) 303-314; M. Yamashita, R. Huba, H.D. Hofmann, Early in vitro development of voltage- and transmitter-gated currents in GABAergic amacrine cells, Dev. Brain Res. 82 (1994) 95-102; R. Ientile, S. Pedale, V. Picciurro, V. Macaione, C. Fabiano, S. Macaione, Nitric oxide mediates NMDA-evoked [3H]GABA release from chick retina cells, FEBS Lett. 417 (1997) 345-348; R.C. Kubrusly, M.C. deMello, F.G. deMello, Aspartate as a selective NMDA agonist in cultured cells from the avian retina, Neurochem. Intl. 32 (1998) 47-52] or reduction of GABA in vivo [N.N. Osborn, A.J. Herrera, The effect of experimental ischaemia and excitatory amino acid agonist on the GABA and serotonin immunoreactivities in the rabbit retina, Neurosci. 59 (1994) 1071-1081]. Since the majority of GABAergic synapses in the inner retina are onto both rod and cone bipolar axon terminals [R.G. Pourcho, M.T. Owzcarzak, Distribution of GABA immunoreactivity in the cat retina: A light and electron-microscopic study, Vis. Neurosci. 2 (1989) 425-435], we hypothesize that the NMDA-receptor plays a crucial role in providing feedback inhibition onto rod and cone bipolar cells.     

Localization of guanylate cyclase-activating protein 2 in mammalian retinas

Guanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca2+, a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC in bovine rod outer segment homogenates. These results suggest that although GCAP1 is involved in the Ca2+-sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons.     

Nitric oxide modulates cGMP levels in neurons of the inner and outer retina in opposite ways

In the mammalian retina, neuronal nitric oxide synthase (NOS) is mainly localized in subpopulations of amacrine cells. One function of nitric oxide (NO) is to stimulate soluble guanylate cyclases which in turn synthesize cGMP. We used an antibody specific for cGMP to demonstrate cGMP-like immunoreactivity (cG-IR) in bovine, rat, and rabbit retinae and investigated the effects on cGMP levels of both exogenously applied NO and of endogenously released NO. We found that cGMP levels in inner and outer retina were controlled in opposite ways. In the presence of the NO-donors SNP, SIN-1 or SNAP, cG-IR was prominent in neurons of the inner retina, mainly in cone bipolar cells, some amacrine and ganglion cells. Retinae incubated in IBMX showed weak cG-IR in bipolar cells. Glutamate increased cG-IR in the inner retina, presumably by stimulating endogenous NO release, whereas NOS inhibitors or GABA and glycine decreased cG-IR in bipolar cells by reducing NO release. In somata, inner segments and spherules of rod photoreceptors the situation was reversed. cG-IR was undetectable in the presence of NO-donors or glutamate, was moderate in IBMX-treated retinae, but increased strongly in the presence of NOS inhibitors or GABA/glycine. We conclude that NO is released endogenously in the retina. In the presence of NO, cGMP levels are increased in neurons of the inner retina, but are decreased in rods.     

Median nerve neuropathy from an old lunate dislocation

We performed a histopathologic and immunohistochemical study of eyes obtained at autopsy of an 84-year-old man from a family with X-linked cone degeneration in which affected members have a 6.5-kilobase deletion in the red cone pigment gene. At his most recent ocular examination, at age 71 years, this patient had had a visual acuity of 20/200 OU, fundus changes suggestive of macular degeneration, borderline-normal full-field rod electroretinograms, and profoundly reduced full-field cone electroretinograms. Histopathologic examination demonstrated marked loss of cone and rod photoreceptors and the retinal pigment epithelium in the central macula. The peripheral cone population was reduced, while the peripheral rod population was relatively preserved. Immunohistochemical examination with an antibody to both red and green cone opsin and an antibody to blue cone opsin disclosed a prominent loss of the red and green cone population and preservation of the blue cone population. These findings show that a defect in the red cone pigment gene can result in extensive degeneration of the red and green cone population across the retina.     

Identification and distribution of photoreceptor subtypes in the neotenic tiger salamander retina

The neotenic tiger salamander retina is a major model system for the study of retinal physiology and circuitry, yet there are unresolved issues regarding the organization of the photoreceptors and the photoreceptor mosaic. The rod and cone subtypes in the salamander retina were identified using a combination of morphological and immunocytochemical markers for specific rod and cone opsin epitopes. Because the visual pigment mechanisms present in the tiger salamander retina are well characterized and the antibodies employed in these studies are specific for particular rod and cone opsin epitopes, we also were able to identify the spectral class of the various rod and cone subtypes. Two classes of rods corresponding to the "red" and "green" rods previously reported in amphibian retinas were identified. In serial semithin section analyses, rods and cones comprised 62.4+/-1.4% and 37.6+/-1.4% of all photoreceptors, respectively. One rod type comprising 98.0+/-0.7% of all rods showed the immunological and morphological characteristics of "red" rods, which are maximally sensitive to middle wavelengths. The second rod subtype comprised 2.0+/-0.7% of all rods and possessed the immunological and morphological characteristics of "green" rods, which are maximally sensitive to short wavelengths. By morphology four cone types were identified, showing three distinct immunological signatures. Most cones (84.8+/-1.5% of all cones), including most large single cones, the accessory and principal members of the double cone, and some small single cones, showed immunolabeling by antisera that recognize long wavelength-sensitive cone opsins. A subpopulation of small single cones (8.4+/-1.7% of all cones) showed immunolabeling for short wavelength-sensitive cone opsin. A separate subpopulation of single cones which included both large and small types (6.8+/-1.4% of all cones) was identified as the UV-Cone population and showed immunolabeling by antibodies that recognize rod opsin epitopes. Analysis of flatmounted retinas yielded similar results. All photoreceptor types appeared to be distributed in all retinal regions. There was no obvious crystalline organization of the various photoreceptor subtypes in the photoreceptor mosaic.     

cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C beta 4 (PLCB4)

Defects in the Drosophila norpA (no receptor potential A) gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4, is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, we isolated several overlapping cDNA clones from a human retina library. The composite cDNA sequence predicts a human PLC beta 4 polypeptide of 1022 amino acid residues (MW 117,000). This PLC beta 4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLC beta 4 monospecific antibody detected a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by fluorescence in situ hybridization.     

Histopathology of the human retina in retinitis pigmentosa

The term, 'retinitis pigmentosa', refers to a heterogeneous group of inherited diseases that cause degeneration of rod and cone photoreceptors in the human retina. Loss of photoreceptor cells is usually followed by alterations in the retinal pigment epithelium and retinal glia. Ultimately, degenerative changes occur in the inner retinal neurons, blood vessels, and optic nerve head. This chapter provides background information on the genetics of retinitis pigmentosa and a summary of the histopathologic alterations in human retinas caused by this disease. Detailed information is provided on the effects of the primary disease process on the rod photoreceptors and changes in the other retinal components, all of which are important considerations for understanding and developing therapies for retinitis pigmentosa.     

Circadian clock functions localized in xenopus retinal photoreceptors

A circadian oscillator that regulates visual function is located somewhere within the vertebrate eye. To determine whether circadian rhythmicity is generated by retinal photoreceptors, we isolated and cultured photoreceptor layers from Xenopus retina. On average, 94% of the viable cells in these preparations were rod or cone photoreceptors. Photoreceptor layers produced melatonin rhythmically, with an average period of 24.3 hr, in constant darkness. The phase of the melatonin rhythm was reset by in vitro exposure of the photoreceptor layers to cycles of either light or quinpirole, a D2 dopamine receptor agonist. These data indicate that other parts of the eye are not necessary for generation or entrainment of retinal circadian melatonin rhythms and suggest that rod and/or cone photoreceptors are circadian clock cells.     

An outbreak of hantavirus pulmonary syndrome, Chile, 1997

A novel splice variant of RGS 9 was isolated from a rat hypothalamus, human retina, and a human kidney (Wilm's) tumor. This variant, termed RGS 9L, differs from the retinal form (termed RGS 9S) identified previously in that it contains a 211- (rat) or 205- (human) amino acid proline-rich domain on the carboxyl terminus. The pattern of RGS 9 mRNA splicing was tissue specific, with striatum, hypothalamus- and nucleus accumbens expressing RGS 9L, whereas retina and pineal expressed RGS 9S almost exclusively. This pattern of mRNA splicing seemed to be highly conserved between human and rodents, suggesting cell-specific differences in the function of these variants. Transient expression of RGS 9L augmented basal and beta-adrenergic receptor-stimulated adenylyl cyclase activity while suppressing dopamine D2 receptor-mediated inhibition. Furthermore, RGS 9L expression greatly accelerated the decay of dopamine D2 receptor-induced GIRK current. These results indicate RGS 9L inhibits heterotrimeric Gi function in vivo, probably by acting as a GTPase-activating protein. The human RGS 9 gene was localized to chromosome 17 q23-24 by radiation hybrid and fluorescent in situ hybridization analyses. The RGS 9 gene is within a previously defined locus for retinitis pigmentosa (RP 17), a disease that has been linked to genes in the rhodopsin/transducin/cGMP signaling pathway.     

Extraction of RNA from single frozen sections

PURPOSE: Recently, mutations in several genes have been identified as being responsible for the pathogenesis of autosomal recessive retinitis pigmentosa (arRP). These genes include rhodopsin, beta-subunit of rod cGMP phosphodiesterase (PDEB), alpha-subunit of rod cGMP phosphodiesterase (PDEA), and alpha-subunit of rod cGMP-gated channel. We here attempted to identify a novel mutation in the PDEB gene in Japanese arRP patients. METHODS: Using the PCR-SSCP method, sequencing analysis, and restriction endonuclease digestion assay, we analyzed the PDEB gene in 17 Japanese families with non-dominant retinitis pigmentosa. RESULTS: A novel Ile535Asn mutation was identified in two patients in a single family and the mutation cosegregated with RP in this family. Among 90 unrelated healthy individuals, no one was identified as homozygous for this mutation, except for one individual who was found to be heterozygous. CONCLUSIONS: Isoleucine at codon 535 in the PDEB gene is conserved among various mammals. Missense mutations of the PDEB gene causing arRP have been reported in a limited region (codon 527-codon 699) in which codon 535 is located. Thus, the Ile535Asn mutation is an additional missense mutation which is responsible for the pathogenesis of arRP.     

The Bacillus SpoIIGA protein is targeted to sites of spore septum formation in a SpoIIE-independent manner

Parvocellular (P-) and magnocellular (M-) cells in the marmoset LGN can receive prominent rod input up to relatively high illuminance levels (Kremers et al., 1997b). In the present paper, we quantify rod and cone input strengths under different retinal illuminance levels. The stimulus was based on the so-called "silent substitution" method. The activities of P- and M-cells of dichromatic animals were recorded extracellularly. We were able to adequately describe the response amplitudes and phases by a vector summation of rod and cone signals. At low retinal illuminance levels, the cells' responses were determined by rod and cone inputs. With increasing illuminances the strength of the cone input increased relative to the rod strength. But, we often found significant rod inputs up to illuminances equivalent to 700 td in the human eye or more. Rod input strength was more pronounced in cells with receptive fields at large retinal eccentricities. The phase differences between rod and cone inputs suggest that the rod signals lag about 45 ms behind the cone signals.     

A case of leiomyosarcoma of the small bowel mesenterium

Kinesins are a large superfamily of microtubule motors that mediate specific motile processes. In a previous study, we identified 11 kinesin family members in the retina and retinal pigment epithelium (RPE) of the striped bass, Morone saxatilus. We have now identified, cloned, and sequenced the human homologue (KIFC3) of the most abundantly expressed retinal kinesin from that study, the C-terminal kinesin FKIF2. An antibody raised against an FKIF2 peptide cross-reacted with an approximately 80-kDa protein in human retina, RPE, kidney, and lung. Since microtubule-dependent processes are critical to the function and morphogenesis of the photoreceptors and RPE, the abundantly expressed KIFC3 was considered to be a potential candidate gene for causing human retinal degeneration. Chromosomal localization of the KIFC3 gene revealed that it maps to chromosome 16q13-q21, within the critical region for a Bardet-Biedl syndrome locus (BBS2). Bardet-Biedl syndrome is a genetically heterogeneous, autosomal recessive disorder characterized by retinal dystrophy, polydactyly, obesity, hypogonadism, renal abnormalities, and mental retardation. The chromosomal localization and expression pattern of KIFC3 suggest that it may be an excellent candidate for families linked to BBS2.     

Absence of short-wavelength sensitive cones in the retinae of seals (Carnivora) and African giant rats (Rodentia)

Most non-primate mammals have two types of cone: short-wavelength sensitive (S) and middle-to-long-wavelength sensitive (M/L) cones. In two species of African giant rats, Cricetomys gambianus and C. emini, and in two species of earless seals, Phoca hispida and P. vitulina, the retinal cone types and cone distributions were assessed with antibodies specific for the M/L-cone opsin and the S-cone opsin, respectively. All four species were found to completely lack S-cones, while M/L-cones were present in low densities. M/L-cone densities, rod densities and cone/rod ratios were determined across the retina. Cone proportions are about 0.3-0. 5% in C. gambianus, 0.5-0.8% in C. emini, and 1.5-1.8% in P. hispida. An absence of S-cones has previously been reported in a few nocturnal mammals. As earless seals are visually active during night and day, we conclude that an absence of S-cones is not exclusively associated with nocturnality. The functional and comparative aspects are discussed.     

Differential localization and expression of alpha and beta isoenzymes of protein kinase C in the rat retina

Expression and cellular localization of three isoenzymes of Ca2+-dependent protein kinase C (PKCalpha, PKCbeta, and PKCgamma) in the adult rat retina were revealed by immunohistochemistry and in situ hybridization histochemistry with isoenzyme-specific antibodies and cRNA probes. Immunoreactivities and mRNA signals for PKCalpha were conspicuous in rod bipolar cells. A subgroup of amacrine cells expressed PKCalpha. The cells in the ganglion cell layer also displayed PKCalpha gene products. Positive immunoreactivities for PKCbeta were localized as stripe patterns in the inner plexiform layer, corresponding to the stratification levels of axon terminals of cone bipolar cells. The somata of cone bipolar cells expressed PKCbeta. Amacrine cells and retinal ganglion cells also displayed PKCbeta gene products. The results obtained by immunohistochemistry were confirmed with colocalization of mRNA signals for PKCalpha and PKCbeta on the somata. The cell membranes showed stronger immunoreactivities than did the cytoplasms for both PKCalpha and PKCbeta. Neither immunoreactivities nor mRNA signals for PKCgamma were detected in all retinal regions. The differential roles of Ca2+-dependent PKC isoenzymes could be revealed in physiological defined retinal neurons.