guanosine-triphosphate and Retinitis-Pigmentosa

Retinitis pigmentosa (RP) is one of the most genetically heterogeneous inherited disorders. Twelve genes have now been identified in the autosomal dominant form of the disease, including some recently characterized genes that show unprecedented and fascinating traits in both their function and in their expression profiles. These include many widely expressed genes encoding components of the spliceosome and a guanine nucleotide synthesis gene. Intriguingly, the most recently identified dominant gene does not appear to be expressed in the neuronal retina but is expressed in the capillaries of the choroid. In attempting to understand the effects of mutations in these genes, investigators are forced to re-evaluate their thinking on the molecular mechanisms of genetic blindness and to undertake an increasingly inter-disciplinary approach in their analysis of this disease. Recently, this has resulted in significant developments in the elucidation of the molecular pathogenesis of RP.

Topics: Animals; Genes, Dominant; Genetic Predisposition to Disease; Guanine; Guanosine Triphosphate; Humans; Light; Models, Biological; Mutation; Protein Folding; Retina; Retinitis Pigmentosa; Rhodopsin; Spliceosomes; Vision, Ocular

The retinitis pigmentosa 2 polypeptide (RP2) functions as a GTPase-activating protein (GAP) for ARL3 (Arf-like protein 3), a small GTPase. ARL3 is an effector of phosphodiesterase 6 Δ (PDE6D), a prenyl-binding protein and chaperone of prenylated protein in photoreceptors. Mutations in the human RP2 gene cause X-linked retinitis pigmentosa (XLRP) and cone-rod dystrophy (XL-CORD). To study mechanisms causing XLRP, we generated an RP2 knockout mouse. The Rp2h(-/-) mice exhibited a slowly progressing rod-cone dystrophy simulating the human disease. Rp2h(-/-) scotopic a-wave and photopic b-wave amplitudes declined at 1 mo of age and continued to decline over the next 6 mo. Prenylated PDE6 subunits and G-protein coupled receptor kinase 1 (GRK1) were unable to traffic effectively to the Rp2h(-/-) outer segments. Mechanistically, absence of RP2 GAP activity increases ARL3-GTP levels, forcing PDE6D to assume a predominantly "closed" conformation that impedes binding of lipids. Lack of interaction disrupts trafficking of PDE6 and GRK1 to their destination, the photoreceptor outer segments. We propose that hyperactivity of ARL3-GTP in RP2 knockout mice and human patients with RP2 null alleles leads to XLRP resembling recessive rod-cone dystrophy.

Topics: ADP-Ribosylation Factors; Animals; Antibody Formation; Caenorhabditis elegans; Cilia; Cyclic Nucleotide Phosphodiesterases, Type 6; Electroretinography; Eye Proteins; Female; G-Protein-Coupled Receptor Kinase 1; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Immunoblotting; Immunoenzyme Techniques; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Photoreceptor Cells, Vertebrate; Protein Prenylation; Protein Transport; Rabbits; Retinitis Pigmentosa

Degenerative retinopathies, including age-related macular degeneration, diabetic retinopathy, and hereditary retinal disorders--major causes of world blindness--are potentially treatable by using low-molecular weight neuroprotective, antiapoptotic, or antineovascular drugs. These agents are, however, not in current systemic use owing to, among other factors, their inability to passively diffuse across the microvasculature of the retina because of the presence of the inner blood-retina barrier (iBRB). Moreover, preclinical assessment of the efficacies of new formulations in the treatment of such conditions is similarly compromised. We describe here an experimental process for RNAi-mediated, size-selective, transient, and reversible modulation of the iBRB in mice to molecules up to 800 Da by suppression of transcripts encoding claudin-5, a protein component of the tight junctions of the inner retinal vasculature. MRI produced no evidence indicative of brain or retinal edema, and the process resulted in minimal disturbance of global transcriptional patterns analyzed in neuronal tissue. We show that visual function can be improved in IMPDH1(-/-) mice, a model of autosomal recessive retinitis pigmentosa, and that the rate of photoreceptor cell death can be reduced in a model of light-induced retinal degeneration by systemic drug delivery after reversible barrier opening. These findings provide a platform for high-throughput drug screening in models of retinal degeneration, and they ultimately could result in the development of a novel "humanized" approach to therapy for conditions with little or no current forms of treatment.

Topics: Animals; Blood-Retinal Barrier; Calpain; Claudin-5; Cysteine Proteinase Inhibitors; Disease Models, Animal; Drug Delivery Systems; Electroretinography; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Magnetic Resonance Imaging; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oligopeptides; Retina; Retinitis Pigmentosa; RNA Interference; RNA, Small Interfering

Retinitis pigmentosa (RP), the hereditary degenerative disease of the photoreceptor neurons of the retina, probably represents the most prevalent cause of registered blindness amongst those of working age in developed countries. Mutations within the gene encoding inosine monophosphate dehydrogenase 1 (IMPDH1), the widely expressed rate-limiting enzyme of the de novo pathway of guanine nucleotide biosynthesis, have recently been shown to cause the RP10 form of autosomal dominant RP. We examined the expression of IMPDH1, IMPDH2 and HPRT transcripts, encoding enzymes of the de novo and salvage pathways of guanine nucleotide biosynthesis, respectively, in retinal sections of mice, the data indicating that the bulk of GTP within photoreceptors is generated by IMPDH1. Impdh1(-/-) null mice are shown here to display a slowly progressive form of retinal degeneration in which visual transduction, analysed by electroretinographic wave functions, becomes gradually compromised, although at 12 months of age most photoreceptors remain structurally intact. In contrast, the human form of RP caused by mutations within the IMPDH1 gene is a severe autosomal dominant degenerative retinopathy in those families that have been examined to date. Expression of mutant IMPDH1 proteins in bacterial and mammalian cells, together with computational simulations, indicate that protein misfolding and aggregation, rather than reduced IMPDH1 enzyme activity, is the likely cause of the severe phenotype experienced by human subjects. Taken together, these findings suggest that RP10 may represent an attractive target for therapeutic intervention, based upon a strategy combining simultaneous suppression of transcripts from normal and mutant IMPDH1 alleles with supplementation of GTP within retinal tissues.

Topics: Animals; Cells, Cultured; Computer Simulation; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Electroretinography; Escherichia coli; Guanine Nucleotides; Guanosine Triphosphate; Histological Techniques; Hypoxanthine Phosphoribosyltransferase; IMP Dehydrogenase; In Situ Hybridization; Mice; Mice, Mutant Strains; Models, Molecular; Protein Folding; Retina; Retinitis Pigmentosa

Opsin phosphorylation in light was detected in three retinas from autopsy eyes with retinitis pigmentosa (RP) including one with sex-linked RP and two with autosomal recessive RP, that were studied at postmortem intervals of 1-4 hr. In these retinas from RP eyes, opsin phosphorylation in light was reduced compared with that in normal human retinas, a finding that is compatible with reduced amounts of opsin due to extensive loss of photoreceptor cells. ATP and GTP levels, although reduced below normal, also were easily detectable, supporting the idea that the reduction in opsin phosphorylation was due to loss of photoreceptor cells and not to a reduced capacity for energy metabolism. These findings in these RP retinas contrast with those in rd mice and Irish setters with rod-cone dysplasia in which a failure of opsin phosphorylation has been detected prior to onset of photoreceptor cell degeneration.

Topics: Adenosine Triphosphate; Adult; Aged; Electrophoresis, Polyacrylamide Gel; Eye Proteins; Guanosine Triphosphate; Humans; Immunoenzyme Techniques; Middle Aged; Phosphorylation; Photoreceptor Cells; Retina; Retinitis Pigmentosa; Rod Opsins