cyclic-gmp and Blindness

Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans.

Topics: Adult; Animals; Blindness; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Cyclic GMP; Fibroblasts; Humans; Induced Pluripotent Stem Cells; Mice; Mutation; Organoids; Orphan Nuclear Receptors; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Skin; Transplantation, Autologous

Substitutions of Arg

Topics: Amino Acid Substitution; Animals; Blindness; Calcium; Calcium Signaling; Cyclic GMP; Disease Models, Animal; Guanylate Cyclase; Humans; Mice; Mice, Transgenic; Mutation, Missense; Receptors, Cell Surface; Retinal Rod Photoreceptor Cells

Defects in the photoreceptor-specific gene encoding aryl hydrocarbon receptor interacting protein like-1 (AIPL1) are linked to blinding diseases, including Leber congenital amaurosis (LCA) and cone dystrophy. While it is apparent that AIPL1 is needed for rod and cone function, the role of AIPL1 in cones is not clear. In this study, using an all-cone animal model lacking Aipl1, we show a light-independent degeneration of M- and S-opsin containing cones that proceeds in a ventral-to-dorsal gradient. Aipl1 is needed for stability, assembly and membrane association of cone PDE6, an enzyme crucial for photoreceptor function and survival. Furthermore, RetGC1, a protein linked to LCA that is needed for cGMP synthesis, was dramatically reduced in cones lacking Aipl1. A defect in RetGC1 is supported by our finding that cones lacking Aipl1 exhibited reduced levels of cGMP. These findings are in contrast to the role of Aipl1 in rods, where destabilization of rod PDE6 results in an increase in cGMP levels, which drives rapid rod degeneration. Our results illustrate mechanistic differences behind the death of rods and cones in retinal degenerative disease caused by deficiencies in AIPL1.

Topics: Adaptor Proteins, Signal Transducing; Animals; Blindness; Catalytic Domain; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Enzyme Stability; Guanylate Cyclase; Humans; Leber Congenital Amaurosis; Mice; Mice, Knockout; Protein Transport; Receptors, Cell Surface; Retina; Retinal Cone Photoreceptor Cells

Recessive mutations in GUCY2D, the gene encoding the retinal guanylyl cyclase protein, RetGC-1, have been shown to cause Leber Congenital Amaurosis (LCA), a severe retinal dystrophy. The purpose of this study was to determine the functional consequences of selected mutations in GUCY2Dlinked to LCA. The mutations investigated in this study map to the catalytic domain (P858S, L954P) and the extracellular domain (C105Y, L325P) of RetGC-1.. All four mutations were introduced into the in vitro expression plasmid, pRC-CMV human RetGC-1, and expressed in HEK-293 cells. We assayed the abilities of the mutant cyclases to generate cGMP (basal activity), and to be activated by guanylyl cyclase activating proteins (GCAP-1 and GCAP-2). Additionally, we co-expressed the catalytic domain mutations (P858S and L954P) with a wild-type allele to test for dominant negative effects on wild-type RetGC-1.. The P858S and L954P mutations, both in highly conserved residues of the catalytic domain of RetGC-1, severely impair basal, GCAP-1, and GCAP-2 stimulated catalytic activity of the enzyme. In addition, when co-expressed with the wild-type allele, both catalytic domain mutations act as dominant negative proteins and reduce the activity of wild-type RetGC-1. The basal activities of the C105Y and L325P mutants are unaltered, but GCAP-1 and GCAP-2 stimulated cyclase activities are reduced approximately 50%.. GUCY2D mutations from LCA patients have distinct functional consequences on RetGC-1 catalytic activity in vitro. Our analyses showed that the catalytic domain mutations cause a marked reduction in cyclase activity, while the extracellular domain mutations moderately reduce activity. The catalytic domain mutant alleles cause dominant negative effects, indicating that the functionality of RetGC-1 is compromised even in heterozygotes. This is consistent with abnormalities in cone electroretinograms (ERGs) detected in obligate heterozygous GUCY2D parents that carry the L954P mutation.

Topics: Alleles; Blindness; Blotting, Western; Calcium-Binding Proteins; Catalytic Domain; Cell Line; Cyclic GMP; Genes, Dominant; Genes, Recessive; Guanylate Cyclase; Guanylate Cyclase-Activating Proteins; Humans; Kidney; Point Mutation; Protein Structure, Tertiary; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Retinal Degeneration; Transfection

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies. It is a genetically heterogeneous condition as six disease-causing genes have been hitherto identified. Among them, RETGC1 (GUCY2D), is more frequently implicated in our series of LCA patients. Interestingly, 70 % of the families with RETGC1 mutations are originating from Mediterranean countries, the remaining families (30%) being originating from various countries across the world. Here, we report, the identification of the same homozygous RETGC1 nonsense mutation in three unrelated and non-consanguineous LCA families of Finnish origin, suggesting a founder effect. Interestingly, no linkage desequilibrium was found using polymorphic markers flanking the RETGC1 gene, supporting the view that the mutation is very ancient. Haplotype studies and Bayesian calculation point the founder mutation to 150 generations (95% credible interval 80-240 generations), i.e., 3000 years ago.

Topics: Blindness; Cyclic GMP; Female; Finland; Founder Effect; Guanine; Guanylate Cyclase; Humans; Linkage Disequilibrium; Male; Mutation; Nuclear Family; Optic Atrophies, Hereditary; Pedigree; Polymorphism, Single Nucleotide; Sequence Deletion

Leber congenital amaurosis (LCA) is the earliest and the most severe form of all inherited retinal dystrophies. In 1996, the current investigators ascribed the disease in families linked to the LCA1 locus on chromosome 17p13.1 to mutations in the photoreceptor-specific guanylyl cyclase (retGC-1) gene. So far, 22 different mutations, of which 11 are missense mutations, have been identified in 25 unrelated families. This is a report of the functional analyses of nine of the missense mutations.. cDNA constructs were generated that contained the retGC-1 missense mutations identified in patients related to the LCA1 locus. Mutants were expressed in COS7 cells and assayed for their ability to hydrolyze guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP).. All mutations lying in the catalytic domain showed a complete abolition of cyclase activity. In contrast, only one mutation lying in the extracellular domain also resulted in a severely reduced catalytic activity, whereas the others showed completely normal activity.. More than half the mutations identified in patients related to the LCA1 locus are truncating mutations expected to result in a total abolition of retGC-1 activity. Concerning missense mutations, half of them lying in the catalytic domain of the protein also result in the complete inability of the mutant cyclases to hydrolyze GTP into cGMP in vitro. In contrast, missense mutations lying in the extracellular domain, except one affecting the initiation codon, showed normal catalytic activity of retGC-1. Nevertheless, considering that all patients related to the LCA1 locus displayed the same phenotype, it can be assumed that all missense mutations would have the same dramatic consequences on protein activity in vivo as truncation mutations.

Topics: Animals; Blindness; Chromosomes, Human, Pair 17; COS Cells; Cyclic GMP; DNA Mutational Analysis; Genetic Linkage; Guanosine Triphosphate; Guanylate Cyclase; Humans; Hydrolysis; Mutation, Missense; Optic Atrophies, Hereditary; Receptors, Cell Surface; Rod Cell Outer Segment

The purpose of this study was to determine if the absence of guanylate cyclase-1 (RetGC1, GC1), a key visual phototransduction cascade enzyme that is expressed in both retinal photoreceptors and pinealocytes, disrupts light regulation of pinopsin mRNA levels in the chicken pineal gland. In this series of experiments, we compared levels of pinopsin and tryptophan 5-hydroxylase mRNA in the pineal glands of GUCY1*B (*B) and normal chickens housed under either cyclic light or constant dark conditions. The *B chicken carries a null mutation in the gene encoding guanylate cyclase-1 that results in blindness in these animals at hatching. The results of our experiments show (1) that the amount of pinopsin mRNA in *B pineal is significantly higher than the amount in normal pineal in both light and dark conditions, (2) that light induces an increase in pinopsin mRNA levels in *B pineal, (3) that the relative magnitude of the light-induced increase in pinopsin mRNA in *B pineal is not significantly different from that observed in normal pineal, and (4) that the changes in the regulation of pinopsin mRNA levels in *B pineal gland are not accompanied by changes in the circadian expression of tryptophan 5-hydroxylase mRNA. These results show that the absence of guanylate cyclase-1 expression in the *B pineal gland leads to a significant increase in basal levels of pinopsin mRNA in this gland but does not alter the magnitude of the increase in pinopsin mRNA levels that is observed as a result of light stimulation.

Topics: Animals; Avian Proteins; Blindness; Chickens; Circadian Rhythm; Crosses, Genetic; Cyclic GMP; Darkness; Gene Deletion; Gene Expression Regulation; Guanylate Cyclase; Light; Nerve Tissue Proteins; Pineal Gland; Receptors, Cell Surface; RNA, Messenger; Rod Opsins; Second Messenger Systems; Tryptophan Hydroxylase

Leber's congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies responsible for congenital blindness. Genetic heterogeneity of LCA has been suspected since the report by Waardenburg of normal children born to affected parents. In 1995 we localised the first disease causing gene, LCA1, to chromosome 17p13 and confirmed the genetic heterogeneity. In 1996 we ascribed LCA1 to mutations in the photoreceptor-specific guanylate cyclase gene (retGC1). Here, we report on the screening of the whole coding sequence of the retGC1 gene in 118 patients affected with LCA. We found 22 different mutations in 24 unrelated families originating from various countries of the world. It is worth noting that all retGC1 mutations consistently caused congenital cone-rod dystrophy in our series, confirming the previous genotype-phenotype correlations we were able to establish. RetGC1 is an essential protein implicated in the phototransduction cascade, especially in the recovery of the dark state after the excitation process of photoreceptor cells by light stimulation. We postulate that the retGC1 mutations hinder the restoration of the basal level of cGMP of cone and rod photoreceptor cells, leading to a situation equivalent to consistent light exposure during photoreceptor development, explaining the severity of the visual disorder at birth.

Topics: Blindness; Chromosomes, Human, Pair 17; Cyclic GMP; Female; Genetic Heterogeneity; Genotype; Guanylate Cyclase; Humans; Male; Mutation; Optic Atrophies, Hereditary; Pedigree; Phenotype; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Receptors, Cell Surface; Rod Cell Outer Segment; Sequence Analysis, DNA

The retinas of the retinal degeneration (rd) chicken are fully developed and possess normal morphology at hatching but fail to respond to light stimulation. Analyses of retinal cGMP, the internal messenger of phototransduction, show that the amount of cGMP in predegenerate, fully developed rd/rd photoreceptors is 5-10 times less than that seen in normal photoreceptor cells. We show that the low levels of cGMP in rd chicken retina are a consequence of a null mutation in the photoreceptor guanylate cyclase (GC1) gene. Thus, the rd chicken is a model for human Leber's congenital amaurosis. Absence of GC1 in rd retina prevents phototransduction and affects survival of rods and cones but does not interfere with normal photoreceptor development.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blindness; Calcium-Binding Proteins; Chickens; Cloning, Molecular; Cyclic GMP; Disease Models, Animal; Down-Regulation; Frameshift Mutation; Gene Rearrangement; Guanylate Cyclase; Guanylate Cyclase-Activating Proteins; Humans; Molecular Sequence Data; Optic Atrophies, Hereditary; Phenotype; Photoreceptor Cells; Receptors, Cell Surface; Retinal Degeneration; Vision, Ocular

Leber's congenital amaurosis (LCA, MIM 204,000), the earliest and most severe form of inherited retinopathy, accounts for at least 5% of all inherited retinal dystrophies. This autosomal recessive condition is usually recognized at birth or during the first months of life in an infant with total blindness or greatly impaired vision, normal fundus and extinguished electroretinogram (ERG). Nystagmus (pendular type) and characteristic eye poking are frequently observed in the first months of life (digito-ocular sign of Franceschetti). Hypermetropia and keratoconus frequently develop in the course of the disease. The observation by Waardenburg of normal children born to affected parents supports the genetic heterogeneity of LCA. Until now, however, little was known about the pathophysiology of the disease, but LCA is usually regarded as the consequence of either impaired development of photoreceptors or extremely early degeneration of cells that have developed normally. We have recently mapped a gene for LCA to chromosome 17p13.1 (LCA1) by homozygosity mapping in consanguineous families of North African origin and provided evidence of genetic heterogeneity in our sample, as LCA1 accounted for 8/15 LCA families in our series. Here, we report two missense mutations (F589S) and two frameshift mutations (nt 460 del C, nt 693 del C) of the retinal guanylate cyclase (RETGC, GDB symbol GUC2D) gene in four unrelated LCA1 probands of North African ancestry and ascribe LCA1 to an impaired production of cGMP in the retina, with permanent closure of cGMP-gated cation channels.

Topics: Blindness; Chromosomes, Human, Pair 17; Cyclic GMP; Frameshift Mutation; Guanylate Cyclase; Homozygote; Humans; Molecular Sequence Data; Mutation; Optic Atrophies, Hereditary; Photoreceptor Cells; Restriction Mapping; Retina