cyclic-gmp and Retinal-Degeneration

Programmed cell death (PCD) is a highly regulated process that results in the orderly destruction of a cell. Many different forms of PCD may be distinguished, including apoptosis, PARthanatos, and cGMP-dependent cell death. Misregulation of PCD mechanisms may be the underlying cause of neurodegenerative diseases of the retina, including hereditary retinal degeneration (RD). RD relates to a group of diseases that affect photoreceptors and that are triggered by gene mutations that are often well known nowadays. Nevertheless, the cellular mechanisms of PCD triggered by disease-causing mutations are still poorly understood, and RD is mostly still untreatable. While investigations into the neurodegenerative mechanisms of RD have focused on apoptosis in the past two decades, recent evidence suggests a predominance of non-apoptotic processes as causative mechanisms. Research into these mechanisms carries the hope that the knowledge created can eventually be used to design targeted treatments to prevent photoreceptor loss. Hence, in this review, we summarize studies on PCD in RD, including on apoptosis, PARthanatos, and cGMP-dependent cell death. Then, we focus on a possible interplay between these mechanisms, covering cGMP-signaling targets, overactivation of poly(ADP-ribose)polymerase (PARP), energy depletion, Ca

Topics: Animals; Cyclic GMP; Humans; Models, Biological; Parthanatos; Photoreceptor Cells; Poly(ADP-ribose) Polymerases; Regulated Cell Death; Retinal Degeneration; Signal Transduction

The second messengers, cGMP and Ca

Topics: Animals; Calcium Signaling; Cell Death; Cyclic GMP; Drug Delivery Systems; Drug Implants; Humans; Nanoparticles; Photoreceptor Cells; Retinal Degeneration

Many RD-causing mutations lead to a dysregulation of cyclic guanosine monophosphate (cGMP), making cGMP signalling a prime target for the development of new treatment approaches. We showed previously that an analogue of cGMP, which inhibited cGMP signalling targets, increased photoreceptor viability in three rodent RD models carrying different genetic defects, in different RD genes. This raises the question of the possible generality of this approach as a treatment for RD. Here, we review RD genes that can be associated with high cGMP and discuss which RD genes might be amenable to a treatment aimed at inhibiting excessive cGMP signalling.

Topics: Animals; Cyclic GMP; Mutation; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Signal Transduction

Hereditary retinal degenerative diseases are mostly diseases of the photoreceptors and/or the retinal pigment epithelium, which lead to loss of vision or even complete blindness. To this day, these diseases are mostly untreatable and represent a considerable burden for patients and their relatives. This review article highlights some of the challenges that arise in the development of new therapies for inherited retinal degeneration, in particular the problem of the enormous genetic heterogeneity of these diseases and the question of how new forms of treatment can be made to cross the blood retinal barrier to the nerve cells of the retina. In this context, the central role of the messenger substance cyclic guanosine mono-phosphate (cGMP) in the photoreceptor is discussed and how this can be used to develop mutation-independent therapies. The DRUGSFORD project will be used as an example to explain how new drugs can be formulated to overcome the blood retinal barrier. In addition, other difficulties will be discussed that arise when positive results from applied research are to be transferred into clinical development. On the one hand, gaps and a lack of interdisciplinarity in the training of scientists and physicians are pointed out; on the other hand, lack of robust data on the natural progression of these disorders and suitable biomarkers also impede clinical development.. Erbliche Netzhautdegenerationen sind Erkrankungen der Photorezeptoren und/oder des Pigmentepithels, die zu einem weitgehenden Sehverlust bis hin zur vollständigen Erblindung führen. Bis heute sind diese seltenen Erkrankungen der Netzhaut zumeist nicht behandelbar und stellen eine erhebliche Belastung für die Betroffenen und ihre Angehörigen dar. Dieser Übersichtsartikel zeigt einige der Herausforderungen auf, die bei der Entwicklung von neuen Therapien für erbliche Netzhautdegenerationen auftreten, insbesondere wird auf das Problem der enormen genetischen Heterogenität dieser Erkrankungen eingegangen und die Frage, wie neue Behandlungsformen über die Blut-Netzhaut-Schranke zu den Nervenzellen der Netzhaut gebracht werden können. In diesem Rahmen wird die zentrale Rolle des Botenstoffs zyklisches Guanosinmonophosphat (cGMP) im Photorezeptor erklärt und wie dessen Funktion genutzt werden kann, um mutationsunabhängige Therapien zu entwickeln. Dies wird am Beispiel des DRUGSFORD-Projektes weiter ausgeführt, wobei auch darauf eingegangen wird, wie neue Medikamente formuliert werden können, um die Blut-Netzhaut-Schranke zu überwinden. Darüber hinaus werden weitere Schwierigkeiten diskutiert, die entstehen, wenn positive Ergebnisse aus der anwendungsbezogenen Forschung in die klinische Entwicklung überführt werden sollen. Hier wird zum einen auf Lücken und mangelnde Interdisziplinarität in der Ausbildung von Naturwissenschaftlern und Medizinern hingewiesen, zum anderen behindert aber auch das Fehlen von Verlaufsdaten und geeigneten Biomarkern die klinische Entwicklung.

Topics: Cyclic GMP; Guanosine; Humans; Neurodegenerative Diseases; Phosphates; Retina; Retinal Degeneration

Photoreceptor physiology and pathophysiology is intricately linked to guanosine-3',5'-cyclic monophosphate (cGMP)-signaling. Here, we discuss the importance of cGMP-signaling for the pathogenesis of hereditary retinal degeneration. Excessive accumulation of cGMP in photoreceptors is a common denominator in cell death caused by a variety of different gene mutations. The cGMP-dependent cell death pathway may be targeted for the treatment of inherited photoreceptor degeneration, using specifically designed and formulated inhibitory cGMP analogues. Moreover, cGMP-signaling and its down-stream targets may be exploited for the development of novel biomarkers that could facilitate monitoring of disease progression and reveal the response to treatment in future clinical trials. We then briefly present the importance of appropriate formulations for delivery to the retina, both for drug and biomarker applications. Finally, the review touches on important aspects of future clinical translation, highlighting the need for interdisciplinary cooperation of researchers from a diverse range of fields.

Topics: Cell Death; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Humans; Photoreceptor Cells; Retina; Retinal Degeneration; Signal Transduction

Humans with mutations in the phototransduction pathway develop forms of retinal degeneration, such as retinitis pigmentosa, cone dystrophy, or Leber congenital amaurosis. Similarly, numerous phototransduction mutant animal models resemble retinal degeneration. In our lab, using a zebrafish model, we study cone-specific phototransduction mutants. cGMP is the second messenger in the phototransduction pathway, and abnormal cGMP levels are associated with photoreceptor death. Rd1, a rod-specific phosphodiesterase 6 (Pde6) subunit mutant in mice, is one of the most widely used animal models for retinal degeneration. Rd1 mutant mice accumulate cGMP, causing rapid photoreceptor degeneration. However, much less is known about photoreceptor mutants producing abnormally low levels of cGMP. Here, focusing on Pde6 mutants in zebrafish and mice, we propose a correlation between cGMP levels and speed of photoreceptor degeneration.

Topics: Animals; Color Vision Defects; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Eye Proteins; Forecasting; Humans; Light Signal Transduction; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Zebrafish; Zebrafish Proteins

Zebrafish are an excellent animal model for research on vertebrate development and human diseases. Sophisticated genetic tools including large-scale mutagenesis methodology make zebrafish useful for studying neuronal degenerative diseases. Here, we review zebrafish models of inherited ophthalmic diseases, focusing on cGMP metabolism in photoreceptors. cGMP is the second messenger of phototransduction, and abnormal cGMP levels are associated with photoreceptor death. cGMP concentration represents a balance between cGMP phosphodiesterase 6 (PDE6) and guanylate cyclase (GC) activities in photoreceptors. Various zebrafish cGMP metabolism mutants were used to clarify molecular mechanisms by which dysfunctions in this pathway trigger photoreceptor degeneration. Here, we review the history of research on the retinal degeneration (rd) mutant mouse, which carries a genetic mutation of PDE6b, and we also highlight recent research in photoreceptor degeneration using zebrafish models. Several recent discoveries that provide insight into cGMP toxicity in photoreceptors are discussed.

Topics: Animals; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Humans; Mice; Mice, Mutant Strains; Mutation; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Vision, Ocular; Zebrafish

Topics: Animals; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Ion Channels; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Vision, Ocular

Cyclic GMP is essential for the ability of rods and cones to respond to the light stimuli. Light triggers hydrolysis of cGMP and stops the influx of sodium and calcium through the cGMP-gated ion channels. The consequence of this event is 2-fold: first, the decrease in the inward sodium current plays the major role in an abrupt hyperpolarization of the cellular membrane; secondly, the decrease in the Ca2+ influx diminishes the free intracellular Ca2+ concentration. While the former constitutes the essence of the phototransduction pathway in rods and cones, the latter gives rise to a potent feedback mechanism that accelerates photoreceptor recovery and adaptation to background light. One of the most important events by which Ca2+ feedback controls recovery and light adaptation is synthesis of cGMP by guanylyl cyclase. Two isozymes of membrane photoreceptor guanylyl cyclase (retGC) have been identified in rods and cones that are regulated by Ca2+-binding proteins, GCAPs. At low intracellular concentrations of Ca2+ typical for light-adapted rods and cones GCAPs activate RetGC, but concentrations above 500 nM typical for dark-adapted photoreceptors turn them into inhibitors of retGC. A variety of mutations found in GCAP and retGC genes have been linked to several forms of human congenital retinal diseases, such as dominant cone degeneration, cone-rod dystrophy and Leber congenital amaurosis.

Topics: Animals; Calcium; Calcium-Binding Proteins; Cyclic GMP; Guanylate Cyclase; Guanylate Cyclase-Activating Proteins; Humans; Mutation; Photoreceptor Cells, Vertebrate; Retinal Degeneration

Topics: Animals; Apoptosis; Awards and Prizes; Base Sequence; Caspase 1; Cyclic GMP; Cysteine Endopeptidases; Dogs; Mice; Molecular Sequence Data; Ophthalmology; Phosphoric Diester Hydrolases; Retinal Degeneration; Retinal Rod Photoreceptor Cells

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Base Sequence; Cyclic GMP; DNA; Mice; Mice, Mutant Strains; Molecular Sequence Data; Retinal Degeneration

Topics: Animals; Calcium; Cyclic GMP; Heterozygote; Mice; Photic Stimulation; Photoreceptor Cells; Retina; Retinal Degeneration

Topics: Animals; Animals, Newborn; Calmodulin; Cyclic GMP; Disease Models, Animal; Dog Diseases; Dogs; Mice; Mice, Neurologic Mutants; Mutation; Nervous System Malformations; Phosphoric Diester Hydrolases; Photoreceptor Cells; Retina; Retinal Degeneration; Rodent Diseases

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cattle; Cyclic GMP; Dog Diseases; Dogs; Guanosine Triphosphate; Guanylate Cyclase; Hydrolysis; Mice; Mice, Mutant Strains; Phosphoproteins; Photoreceptor Cells; Rabbits; Rats; Retina; Retinal Degeneration; Rod Cell Outer Segment; Rodent Diseases

Topics: Animals; Aspirin; Cortisone; Cyclic AMP; Cyclic GMP; Darkness; DNA; Lysosomes; Mice; Nucleotides, Cyclic; Rats; Rats, Inbred Strains; Retina; Retinal Degeneration; Salicylates; Species Specificity

Hereditary degeneration of photoreceptors has been linked to over-activation of Ca

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Death; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Diltiazem; Ion Channel Gating; Kinetics; Mice; Proteolysis; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells

Catalytic hydrolysis of cyclic guanosine monophosphate (cGMP) by phosphodiesterase 6 (PDE6) is critical in phototransduction signalling in photoreceptors. Mutations in the genes encoding any of the three PDE6 subunits are associated with retinitis pigmentosa, the most common form of inherited retinal diseases. The RD1 mouse carries a naturally occurring nonsense mutation in the Pde6b gene. The RD1 mouse retina rapidly degenerates and fails to form rod photoreceptor outer segments due to the elevated cGMP level and subsequent excessive Ca2+ influx. In this study, we aim to test whether the PDE5 expression, a non-photoreceptor-specific member of the PDE superfamily, rescues photoreceptors in the RD1 retina.. Electroporation used the PDE5 expression plasmid to transfect neonatal RD1 mice. The mouse retina degeneration was assessed by retinal sections' stains with DAPI. The expression and localization of phototransduction proteins in photoreceptors were analysed by immunostaining. The expression of proteins in cultured cells was analysed by immunoblotting.. The exogenous PDE5 expression, a non-photoreceptor-specific member of the PDE superfamily, prevents photoreceptor degeneration in RD1 mice. Unlike endogenous photoreceptor-specific PDE6 localised in the outer segments of photoreceptors, ectopically- expressed PDE5 was distributed in inner segments and synaptic terminals. PDE5 also promoted the development of the outer segments in RD1 mice. PDE5 co-expression with rhodopsin in cultured cells showed enhanced rhodopsin expression.. Lowering the cGMP level in photoreceptors by PDE5 is sufficient to rescue photoreceptors in RD1 retinas. cGMP may also play a role in rhodopsin expression regulation in photoreceptors.

Topics: Animals; Calcium; Codon, Nonsense; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Guanosine Monophosphate; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration; Rhodopsin

Inherited retinal degenerations (IRDs) are a group of blinding diseases, typically involving a progressive loss of photoreceptors. The IRD pathology is often based on an accumulation of cGMP in photoreceptors and associated with the excessive activation of calpain and poly (ADP-ribose) polymerase (PARP). Inhibitors of calpain or PARP have shown promise in preventing photoreceptor cell death, yet the relationship between these enzymes remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type and IRD-mutant mice were treated with inhibitors specific for calpain, PARP, and voltage-gated Ca

Topics: Adenosine Diphosphate; Animals; Calpain; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Mice; Nerve Tissue Proteins; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Retinal Degeneration; Ribose

The disease retinitis pigmentosa (RP) leads to photoreceptor degeneration by a yet undefined mechanism(s). In several RP mouse models (i.e.,

Topics: Animals; Cyclic GMP; Disease Models, Animal; Guanine; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration; Retinitis Pigmentosa

Cyclin dependent kinase 1 (CDK1) has long been known to drive the cell cycle and to regulate the division and differentiation of cells. Apart from its role in mitosis regulation, it also exerts multiple functions as a protein kinase, including engagement in cell death, possibly via a cell cycle-independent mechanism. The latter is suggested, since CDK1 re-expression can be found in non-dividing and terminally differentiated neurons in several neurodegeneration models. However, the details of if and how CDK1 might be involved in the neurodegenerative condition, retinitis pigmentosa (RP), which displays progressive vision loss, are unclear. In the present study, we investigated CDK1 in degenerating RP photoreceptors of the

Topics: Animals; CDC2 Protein Kinase; Cell Death; Cyclic GMP; Mice; Retina; Retinal Degeneration; Retinitis Pigmentosa

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death. Overactivation of the cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) pathway in photoreceptor cells causes photoreceptor cell death, especially in models harboring phosphodiesterase 6b (PDE6b) mutations. Previous studies on RD have used mainly murine models such as rd1 or rd10 mice. Given the genetic and physiological differences between mice and humans, it is important to understand to which extent the retinas of primates and rodents are comparable. Macaques share a high level of genetic similarity with humans. Therefore, wild-type macaques (aged 1-3 years) were selected for the in vitro culture of retinal explants that included the retina-retinal pigment epithelium (RPE)-choroid complex. These explants were treated with different concentrations of the PDE6 inhibitor zaprinast to induce the cGMP-PKG signaling pathway and simulate RD pathogenesis. cGMP accumulation and cell death in primate retinal explants were subsequently verified using immunofluorescence and the TUNEL assay. The primate retinal model established in this study may serve for relevant and effective studies into the mechanisms of cGMP-PKG-dependent RD, as well as for the development of future treatment approaches.

Topics: Animals; Cyclic GMP; Haplorhini; Humans; Macaca; Mice; Retina; Retinal Degeneration

Retinitis Pigmentosa represents a group of genetic disorders that cause progressive vision loss via degeneration of photoreceptors, but there is in principle no treatment available. For any therapy development, a deeper comprehension of the disease-leading mechanism(s) at the molecular level is needed. Here we focused on the cGMP-PKG system, which has been suggested to be a driver in several models of the disease. To gain insights in its downstream signaling we manipulated the cGMP-PKG system with the aid of organotypic retinal explant cultures from either a mouse-based disease model, i.e. the rd1 mouse, or its healthy wild-type counterpart (wt), by adding different types of cGMP analogues to either inhibit or activate PKG in retinal explants from rd1 and wt, respectively. An RNA sequencing was then performed to study the cGMP-PKG dependent transcriptome. Expression changes of gene sets related to specific pathways or functions, that fulfilled criteria involving that the changes should match PKG activation and inhibition, were determined via bioinformatics. The analyses highlighted that several gene sets linked to oxidative phosphorylation and mitochondrial pathways were regulated by this enzyme system. Specifically, the expression of such pathway components was upregulated in the rd1 treated with PKG inhibitor and downregulated in the wt with PKG activator treatment, suggesting that cGMP-PKG act as a negative regulator in this context. Downregulation of energy production pathways may thus play an integral part in the mechanism behind the degeneration for at least several RP mutations.

Topics: Animals; Cells, Cultured; Cyclic GMP; Disease Models, Animal; DNA; DNA Mutational Analysis; Mice; Mice, Inbred C3H; Mutation; Protein Kinases; Retina; Retinal Degeneration; Retinitis Pigmentosa; Signal Transduction; Transcriptome

The hereditary disease Retinitis pigmentosa results in severe vision loss due to photoreceptor degeneration by unclear mechanisms. In several disease models, the second messenger cGMP accumulates in the degenerating photoreceptors, where it may over-activate specific cGMP-interacting proteins, like cGMP-dependent protein kinase. Moreover, interventions that counteract the activity of these proteins lead to reduced photoreceptor cell death. Yet there is little or no information whether other than such regular cGMP-interactors are present in the retina, which we, therefore, investigated in wild-type and retinal degeneration (rd1, rd10, and rd2) mouse models. An affinity chromatography based proteomics approach that utilized immobilized cGMP analogs was applied to enrich and select for regular and potentially new cGMP-interacting proteins as identified by mass spectrometry. This approach revealed 12 regular and 10 potentially new retinal cGMP-interacting proteins (e.g., EPAC2 and CaMKIIα). Several of the latter were found to be expressed in the photoreceptors and to have proximity to cGMP and may thus be of interest when defining prospective therapeutic targets or biomarkers for retinal degeneration.

Topics: Animals; Cyclic GMP; Female; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Proteomics; Retina; Retinal Degeneration

Topics: Amino Acid Sequence; Biomarkers; Carrier Proteins; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Disease Susceptibility; Enzyme Activation; Genetic Predisposition to Disease; Humans; Kinetics; Protein Binding; Retinal Degeneration; Substrate Specificity

Retinitis pigmentosa (RP) is a debilitating blinding disease affecting over 1.5 million people worldwide, but the mechanisms underlying this disease are not well understood. One of the common models used to study RP is the retinal degeneration-10 (rd10) mouse, which has a mutation in Phosphodiesterase-6b (Pde6b) that causes a phenotype mimicking the human disease. In rd10 mice, photoreceptor cell death occurs with exposure to normal light conditions, but as demonstrated in this study, rearing these mice in dark preserves their retinal function. We found that inactivating rhodopsin signaling protected photoreceptors from degeneration suggesting that the pathway activated by this G-protein-coupled receptor is causing light-induced photoreceptor cell death in rd10 mice. However, inhibition of transducin signaling did not prevent the loss of photoreceptors in rd10 mice reared under normal light conditions implying that the degeneration caused by rhodopsin signaling is not mediated through its canonical G-protein transducin. Inexplicably, loss of transducin in rd10 mice also led to photoreceptor cell death in darkness. Furthermore, we found that the rd10 mutation in Pde6b led to a reduction in the assembled PDE6αβγ2 complex, which was corroborated by our data showing mislocalization of the γ subunit. Based on our findings and previous studies, we propose a model where light activates a non-canonical pathway mediated by rhodopsin but independent of transducin that sensitizes cyclic nucleotide gated channels to cGMP and causes photoreceptor cell death. These results generate exciting possibilities for treatment of RP patients without affecting their vision or the canonical phototransduction cascade.

Topics: Animals; Cell Death; cis-trans-Isomerases; Cyclic GMP; Disease Models, Animal; Light; Mice; Mice, Inbred C57BL; Mice, Knockout; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Transducin

Mutations in the

Topics: Animals; Biomarkers; Color Vision Defects; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type II; Cyclic Nucleotide-Gated Cation Channels; Disease Models, Animal; Disease Susceptibility; Endoplasmic Reticulum Stress; Fluorescent Antibody Technique; Gene Expression; Mice; Mice, Knockout; Microscopy, Confocal; Models, Biological; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Unfolded Protein Response

The nitric oxide - guanylyl cyclase-1 - cyclic guanylate monophosphate (NO-GC-1-cGMP) pathway has emerged as a potential pathogenic mechanism for glaucoma, a common intraocular pressure (IOP)-related optic neuropathy characterized by the degeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve. NO activates GC-1 to increase cGMP levels, which are lowered by cGMP-specific phosphodiesterase (PDE) activity. This pathway appears to play a role in both the regulation of IOP, where reduced cGMP levels in mice leads to elevated IOP and subsequent RGC degeneration. Here, we investigated whether potentiation of cGMP signaling could protect RGCs from glaucomatous degeneration. We administered the PDE5 inhibitor tadalafil orally (10 mg/kg/day) in murine models of two forms of glaucoma - primary open angle glaucoma (POAG; GC-1

Topics: Animals; Apoptosis; Cells, Cultured; Cyclic GMP; Disease Models, Animal; Female; Glaucoma; Guanylate Cyclase; Mice, Knockout; Phosphodiesterase 5 Inhibitors; Rats, Sprague-Dawley; Retinal Degeneration; Retinal Ganglion Cells; Signal Transduction; Tadalafil

Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of "druggable" targets, and the access-limiting blood-retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (

Topics: Animals; Blood-Retinal Barrier; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Drug Delivery Systems; Liposomes; Mice; Photoreceptor Cells; Retina; Retinal Degeneration; Signal Transduction

Aryl-hydrocarbon receptor interacting protein-like 1 (AIPL1) is essential to stabilize cGMP phosphodiesterase 6 (PDE6) in rod photoreceptors. Mutation of AIPL1 leads to loss of PDE6, accumulation of intracellular cGMP, and rapid degeneration of rods. To understand the metabolic basis for the photoreceptor degeneration caused by excessive cGMP, we performed proteomics and phosphoproteomics analyses on retinas from AIPL1-/- mice at the onset of rod cell death. AIPL1-/- retinas have about 18 times less than normal PDE6a and no detectable PDE6b. We identified twelve other proteins and thirty-nine phosphorylated proteins related to cell metabolism that are significantly altered preceding the massive degeneration of rods. They include transporters, kinases, phosphatases, transferases, and proteins involved in mitochondrial bioenergetics and metabolism of glucose, lipids, amino acids, nucleotides, and RNA. In AIPLI-/- retinas mTOR and proteins involved in mitochondrial energy production and lipid synthesis are more dephosphorylated, but glycolysis proteins and proteins involved in leucine catabolism are more phosphorylated than in normal retinas. Our findings indicate that elevating cGMP rewires cellular metabolism prior to photoreceptor degeneration and that targeting metabolism may be a productive strategy to prevent or slow retinal degeneration.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Energy Metabolism; Eye Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphoproteins; Phosphorylation; Protein Processing, Post-Translational; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells

Calcium ions play a critical role in neuronal cell death. Pigment epithelium-derived factor (PEDF) is a promising neuroprotective protein for photoreceptor cells but the mechanisms mediating its effects against retinal degeneration are still not well characterized. We addressed this question in the rd1 degenerating mouse retina that bears a mutation in the Pde6b gene encoding one subunit of the phosphodiesterase enzyme. Loss of phosphodiesterase activity in rod photoreceptor cells increases cyclic guanosine monophosphate (cGMP) levels leading to a rise in intracellular calcium. Short-term treatments with recombinant human PEDF protein decreased intracellular calcium in photoreceptors in vivo. Taking advantage of calcium pump blockers, we defined that PEDF signaling acts on PMCA calcium pumps to lower intracellular calcium. PEDF restrained cell death pathways activated by high calcium levels and engaging calpains, BAX and AIF. The neurotrophic effects were mediated by the PEDF receptor (PEDF-R), encoded by the PNPLA2 gene. Finally, peptides containing the neurotrophic domain of PEDF targeted these same cell death pathways in vivo. The findings reveal rescue from death of degenerating photoreceptor cells by a PEDF-mediated preservation of intracellular calcium homeostasis.

Topics: Animals; Calcium; Calcium Signaling; Cell Line; Cyclic GMP; Eye Proteins; Humans; Mice; Mice, Transgenic; Mutation; Nerve Growth Factors; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinal Pigment Epithelium; Serpins

Guanylate Cyclase-Activating Protein 1 (GCAP1) regulates the enzymatic activity of the photoreceptor guanylate cyclases (GC), leading to inhibition or activation of the cyclic guanosine monophosphate (cGMP) synthesis depending on its Ca2+- or Mg2+-loaded state. By genetically screening a family of patients diagnosed with cone-rod dystrophy, we identified a novel missense mutation with autosomal dominant inheritance pattern (c.332A>T; p.(Glu111Val); E111V from now on) in the GUCA1A gene coding for GCAP1. We performed a thorough biochemical and biophysical investigation of wild type (WT) and E111V human GCAP1 by heterologous expression and purification of the recombinant proteins. The E111V substitution disrupts the coordination of the Ca2+ ion in the high-affinity site (EF-hand 3, EF3), thus significantly decreasing the ability of GCAP1 to sense Ca2+ (∼80-fold higher Kdapp compared to WT). Both WT and E111V GCAP1 form dimers independently on the presence of cations, but the E111V Mg2+-bound form is prone to severe aggregation over time. Molecular dynamics simulations suggest a significantly increased flexibility of both the EF3 and EF4 cation binding loops for the Ca2+-bound form of E111V GCAP1, in line with the decreased affinity for Ca2+. In contrast, a more rigid backbone conformation is observed in the Mg2+-bound state compared to the WT, which results in higher thermal stability. Functional assays confirm that E111V GCAP1 interacts with the target GC with a similar apparent affinity (EC50); however, the mutant shifts the GC inhibition out of the physiological [Ca2+] (IC50E111V ∼10 μM), thereby leading to the aberrant constitutive synthesis of cGMP under conditions of dark-adapted photoreceptors.

Topics: Biophysical Phenomena; Calcium; Cone-Rod Dystrophies; Cyclic GMP; Gene Expression Regulation; Guanylate Cyclase-Activating Proteins; Humans; Magnesium; Molecular Dynamics Simulation; Mutation, Missense; Pedigree; Protein Aggregation, Pathological; Protein Binding; Retinal Cone Photoreceptor Cells; Retinal Degeneration

Genetic mutations in aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) cause photoreceptor degeneration associated with Leber congenital amaurosis 4 (LCA4) in human patients. Here we report retinal phenotypes of a zebrafish aipl1 mutant, gold rush (gosh). In zebrafish, there are two aipl1 genes, aipl1a and aipl1b, which are expressed mainly in rods and cones, respectively. The gosh mutant gene encodes cone-specific aipl1, aipl1b. Cone photoreceptors undergo progressive degeneration in the gosh mutant, indicating that aipl1b is required for cone survival. Furthermore, the cone-specific subunit of cGMP phosphodiesterase 6 (Pde6c) is markedly decreased in the gosh mutant, and the gosh mutation genetically interacts with zebrafish pde6c mutation eclipse (els). These data suggest that Aipl1 is required for Pde6c stability and function. In addition to Pde6c, we found that zebrafish cone-specific guanylate cyclase, zGc3, is also decreased in the gosh and els mutants. Furthermore, zGc3 knockdown embryos showed a marked reduction in Pde6c. These observations illustrate the interdependence of cGMP metabolism regulators between Aipl1, Pde6c, and Gc3 in photoreceptors.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Survival; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Epistasis, Genetic; Fertilization; Guanylate Cyclase; Mutation; Opsins; Phenotype; Protein Stability; Protein Transport; Retinal Cone Photoreceptor Cells; Retinal Degeneration; RNA, Messenger; Subcellular Fractions; Zebrafish; Zebrafish Proteins

Cyclic-GMP is a second messenger in phototransduction, a G-protein signaling cascade that conveys photon absorption by rhodopsin to a change in current at the rod photoreceptor outer segment plasma membrane. Basal cGMP level is strictly controlled by the opposing actions of phosphodiesterase (PDE6) and retinal guanylyl cyclases (GCs), and mutations in genes that disrupt cGMP homeostasis leads to retinal degeneration in humans through mechanisms that are incompletely understood. The purpose of this study is to examine two distinct cellular targets of cGMP: the cGMP-gated (CNG) channels and protein kinase G (PRKG), and how each may contribute to rod cell death. Using a mouse genetic approach, we found that abolishing expression of CNG channels prolongs rod survival caused by elevated cGMP in a PDE6 mutant mouse model. This observation supports the use of channel blockers to delay rod death, which is expected to prolong useful vision through enhanced cone survival. However, the absence of CNG channel alone also caused abnormal cGMP accumulation. In a mouse model of CNG channel loss-of-function, abolishing PRKG1 expression had a long-lasting effect in promoting rod cell survival. Our data strongly implicate two distinct cGMP-mediated cell death pathways, and suggest that therapeutic designs targeting both pathways will be more effective at slowing photoreceptor cell death caused by elevated cGMP.

Topics: Animals; Cell Death; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide-Gated Cation Channels; Guanylate Cyclase; Ion Channels; Mice; Mice, Knockout; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Rod Cell Outer Segment; Signal Transduction

Topics: Alleles; Animals; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Disease Models, Animal; Electroretinography; Gene Order; Genetic Loci; Genotype; Mice; Mice, Knockout; Microscopy; Nerve Tissue Proteins; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Tomography, Optical Coherence

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

The enzyme poly-ADP-ribose-polymerase (PARP) mediates DNA-repair and rearrangements of the nuclear chromatin. Generally, PARP activity is thought to promote cell survival and in recent years a number of PARP inhibitors have been clinically developed for cancer treatment. Paradoxically, PARP activity is also connected to many diseases including the untreatable blinding disease Retinitis Pigmentosa (RP), where PARP activity appears to drive the pathogenesis of photoreceptor loss. We tested the efficacy of three different PARP inhibitors to prevent photoreceptor loss in the rd1 mouse model for RP. In retinal explant cultures in vitro, olaparib had strong and long-lasting photoreceptor neuroprotective capacities. We demonstrated target engagement by showing that olaparib reduced photoreceptor accumulation of poly-ADP-ribosylated proteins. Remarkably, olaparib also reduced accumulation of cyclic-guanosine-monophosphate (cGMP), a characteristic marker for photoreceptor degeneration. Moreover, intravitreal injection of olaparib in rd1 animals diminished PARP activity and increased photoreceptor survival, confirming in vivo neuroprotection. This study affirms the role of PARP in inherited retinal degeneration and for the first time shows that a clinically approved PARP inhibitor can prevent photoreceptor degeneration in an RP model. The wealth of human clinical data available for olaparib highlights its strong potential for a rapid clinical translation into a novel RP treatment.

Topics: Animals; Cell Survival; Chromatin; Cyclic GMP; Immunohistochemistry; Mice; Mice, Inbred C3H; Neoplasms; Neuroprotective Agents; Photoreceptor Cells, Vertebrate; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Binding; Quantitative Structure-Activity Relationship; Rabbits; Retinal Degeneration

The duration of cell death may allow deducing the underlying degenerative mechanism. To find out how long a photoreceptor takes to die, we used the rd1 mouse model for retinal neurodegeneration, which is characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cGMP levels. Based on cellular data on the progression of cGMP accumulation, cell death, and survival, we created a mathematical model to simulate the temporal development of the degeneration and the clearance of dead cells. Both cellular data and modelling suggested that at the level of the individual cell, the degenerative process was rather slow, taking around 80 h to complete. Organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast, confirmed the surprisingly long duration of an individual photoreceptor cell's death. We briefly discuss the possibility to link different cell death stages and their temporal progression to specific enzymatic activities known to be causally connected to cell death. This in turn opens up new perspectives for the treatment of inherited retinal degeneration, both in terms of therapeutic targets and temporal windows-of-opportunity.

Topics: Animals; Apoptosis; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide-Gated Cation Channels; Disease Models, Animal; Histone Deacetylases; Mice; Mice, Inbred C3H; Necrosis; Photoreceptor Cells, Vertebrate; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Retinal Degeneration

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca2+-free and Ca2+-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca2+ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca2+. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF-GCAP2), we show that GCAP2 locked in its Ca2+-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca2+-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF-GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca2+-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca2+ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in "equivalent-light" scenarios.

Topics: Animals; Calcium; Calcium Signaling; Calcium-Binding Proteins; Cyclic GMP; EF Hand Motifs; Guanylate Cyclase-Activating Proteins; Humans; Mice; Neurons; Phosphorylation; Photoreceptor Cells; Retina; Retinal Degeneration

Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments.

Topics: Animals; Animals, Genetically Modified; Calpain; Cell Death; Cyclic GMP; Disease Models, Animal; Histone Deacetylases; Light Signal Transduction; Mice; Mutation; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Rats; Retinal Degeneration

Inherited retinal degenerations affecting both rod and cone photoreceptors constitute one of the causes of incurable blindness in the developed world. Cyclic guanosine monophosphate (cGMP) is crucial in the phototransduction and, mutations in genes related to its metabolism are responsible for different retinal dystrophies. cGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4-5% of the retinitis pigmentosa, a rare form of retinal degeneration. The aim of this study was to evaluate whether pharmacological PDE6 inhibition induced retinal degeneration in cone-enriched cultures of porcine retina similar to that found in murine models. PDE6 inhibition was induced in cone-enriched retinal explants from pigs by Zaprinast. PDE6 inhibition induced cGMP accumulation and triggered retinal degeneration, as determined by TUNEL assay. Western blot analysis and immunostaining indicated that degeneration was accompanied by caspase-3, calpain-2 activation and poly (ADP-ribose) accumulation. Oxidative stress markers, total antioxidant capacity, thiobarbituric acid reactive substances (TBARS) and nitric oxide measurements revealed the presence of oxidative damage. Elevated TNF-alpha and IL-6, as determined by enzyme immunoassay, were also found in cone-enriched retinal explants treated with Zaprinast. Our study suggests that this ex vivo model of retinal degeneration in porcine retina could be an alternative model for therapeutic research into the mechanisms of photoreceptor death in cone-related diseases, thus replacing or reducing animal experiments.

Topics: Animals; Apoptosis; Calpain; Caspase 3; Cyclic GMP; In Situ Nick-End Labeling; Organ Culture Techniques; Oxidative Stress; Phosphodiesterase Inhibitors; Purinones; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Swine; Swine, Miniature

Cyclic guanosine 3'-5'-monophosphate (cGMP) plays a key role in the physiological process of light detection in photoreceptor cells of the retina. However, there is also growing evidence that cGMP may be critically involved in some pathophysiological processes of the retina since degenerating photoreceptors in mouse models of retinitis pigmentosa and achromatopsia accumulate high levels of cGMP. Here, we describe methods that allow the detection, subcellular localization, and quantification of cGMP in the retina and propose that cGMP accumulation can be used as a biomarker for photoreceptor degeneration.

Topics: Animals; Cyclic GMP; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique, Indirect; Mice; Retinal Degeneration

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca(2+) influx in rod and cone photoreceptors. cGMP, the native ligand of the photoreceptor CNG channels, has been associated with cytotoxicity when its levels rise above normal due to defects in photoreceptor phosphodiesterase (PDE6) or regulation of retinal guanylyl cyclase (retGC). We found a massive accumulation of cGMP in CNGA3-deficient retina and investigated whether cGMP accumulation plays a role in cone degeneration in CNG channel deficiency. The time course study showed that the retinal cGMP level in Cnga3(-/-);Nrl(-/-) mice with CNGA3 deficiency on a cone-dominant background was sharply increased at postnatal day 8 (P8), peaked around P10-P15, remained high through P30-P60, and returned to near control level at P90. This elevation pattern correlated with photoreceptor apoptotic death, which peaked around P15-P20. In Cnga3(-/-);Gucy2e(-/-) mice lacking retGC1, cone density and expression levels of cone-specific proteins were significantly increased compared with Cnga3(-/-), consistent with a role of cGMP accumulation as the major contributor to cone death caused by CNG channel deficiency. The activity and expression levels of cGMP-dependent protein kinase G (PKG) were significantly increased in Cnga3(-/-);Nrl(-/-) retina compared with Nrl(-/-), suggesting an involvement of PKG regulation in cell death. Our results indicate that cGMP accumulation in photoreceptors can itself exert cytotoxic effect in cones, independently of CNG channel activity and Ca(2+) influx.

Topics: Animals; Animals, Newborn; Carrier Proteins; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide-Gated Cation Channels; Enzyme-Linked Immunosorbent Assay; Eye Proteins; Guanylate Cyclase; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Phosphoric Diester Hydrolases; Photoreceptor Cells; Receptors, Cell Surface; Receptors, N-Methyl-D-Aspartate; Retina; Retinal Degeneration

Two calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) activate cGMP synthesis in photoreceptor by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) to expedite recovery, but calcium-insensitive constitutively active GCAP1 mutants cause photoreceptor degeneration in human patients and transgenic mice. Although GCAP1 and GCAP2 can both activate RetGC1 and RetGC2 in vitro, we find that GCAP1 selectively regulates RetGC1 in vivo. Furthermore, elimination of RetGC1 but not RetGC2 isozyme reverses abnormal calcium sensitivity of cGMP synthesis and rescues mouse rods in transgenic mice expressing GCAP1 mutants causing photoreceptor disease. Rods expressing mutant GCAP1 not only survive in the absence of RetGC1 but also remain functional, albeit with reduced electroretinography (ERG) amplitudes typical of RetGC1-/- genotype. The rod ERG recovery from a strong flash, only slightly affected in both RetGC1-/- and RetGC2-/- mice, becomes very slow in RetGC1-/- but not RetGC2-/- mice when GCAP2 is not available to provide Ca²⁺ feedback to the remaining RetGC isozyme. The intrinsic biochemical properties of RetGC and GCAP determined in vitro do not explain the observed phenomena. Instead, our results argue that there must be a cellular mechanism that limits GCAP1 access to RetGC2 and makes RetGC1 isozyme a preferential target for the disease-causing GCAP1 mutants. A more general conclusion from our findings is that nondiscriminatory interactions between homologous effector enzymes and their regulatory proteins permitted by their intrinsic biochemical properties can be effectively restricted in a living photoreceptor.

Topics: Animals; Calcium; Cyclic GMP; Dose-Response Relationship, Drug; Electroretinography; Female; Guanylate Cyclase; Guanylate Cyclase-Activating Proteins; Isoenzymes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells

Inherited retinal degeneration affecting both rod and cone photoreceptors constitutes one of the leading causes of blindness in the developed world. Such degeneration is at present untreatable, and the underlying neurodegenerative mechanisms are unknown, even though certain genetic causes have been established. The rd1 mouse is one of the best characterized animal models for rod photoreceptor degeneration, whereas the cpfl1 mouse is a recently discovered model for cone cell death. Because both animal models are affected by functionally similar mutations in the rod and cone phosphodiesterase 6 genes, respectively, we asked whether the mechanisms of photoreceptor degeneration in these two mouse lines share common pathways. In the present study, we followed the temporal progression of photoreceptor degeneration in the cpfl1 retina, correlated it with specific metabolic markers, and compared it with the wild-type and the rd1 situation. Similar to corresponding rd1 observations, cpfl1 cone photoreceptor cell death was associated with an accumulation of cyclic guanosine monophosphate (cGMP), activity of calpains, and phosphorylation of vasodilator-stimulated protein (VASP). Cone degeneration progressed rapidly, with a peak in cell death around postnatal day 24. Furthermore, cpfl1 cone photoreceptor migration during early postnatal development was delayed significantly compared with the corresponding wild-type retina. The finding that rod and cone photoreceptor degeneration was associated with the same metabolic markers suggests that in both cell types similar degenerative mechanisms are active. This raises the possibility that equivalent neuroprotective strategies may be used to prevent both rod and cone photoreceptor degeneration.

Topics: Animals; Cell Death; Cell Movement; Cyclic GMP; Disease Models, Animal; Humans; Mice; Mice, Inbred C57BL; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration

Guanylate cyclases, GC1 and GC2, are localized in the light-sensitive outer segment compartment of photoreceptor cells, where they play a crucial role in phototransduction by catalyzing the synthesis of cGMP, the second messenger of phototransduction, and regulating intracellular Ca(2+) levels in combination with the cGMP-gated channel. Mutations in GC1 are known to cause Leber congenital amaurosis type 1 (LCA1), a childhood disease associated with severe vision loss. Although the enzymatic and regulatory properties of guanylate cyclases have been studied extensively, the molecular determinants responsible for their trafficking in photoreceptors remain unknown. Here we show that RD3, a protein of unknown function encoded by a gene associated with photoreceptor degeneration in humans with Leber congenital amaurosis type 12 (LCA12), the rd3 mouse, and rcd2 collie, colocalizes and interacts with GC1 and GC2 in rod and cone photoreceptor cells of normal mice. GC1 and GC2 are undetectable in photoreceptors of the rd3 mouse deficient in RD3 by immunofluorescence microscopy. Cell expression studies show that RD3 mediates the export of GC1 from the endoplasmic reticulum to endosomal vesicles, and that the C terminus of GC1 is required for RD3 binding. Our results indicate that photoreceptor degeneration in the rd3 mouse, rcd2 dog, and LCA12 patients is caused by impaired RD3-mediated guanylate cyclase expression and trafficking. The resulting deficiency in cGMP synthesis and the constitutive closure of cGMP-gated channels might cause a reduction in intracellular Ca(2+) to a level below that required for long-term photoreceptor cell survival.

Topics: Animals; Calcium; Cyclic GMP; Dogs; Guanylate Cyclase; Humans; Leber Congenital Amaurosis; Mice; Mice, Knockout; Nuclear Proteins; Photoreceptor Cells; Protein Transport; Receptors, Cell Surface; Retinal Degeneration

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness in humans. Previously, excessive activation of enzymes belonging to the poly-ADP-ribose polymerase (PARP) group was shown to be involved in photoreceptor degeneration in the human homologous rd1 mouse model for RP. Since there are at least 16 different PARP isoforms, we investigated the exact relevance of the predominant isoform - PARP1 - for photoreceptor cell death using PARP1 knock-out (KO) mice. In vivo and ex vivo morphological analysis using optic coherence tomography (OCT) and conventional histology revealed no major alterations of retinal phenotype when compared to wild-type (wt). Likewise, retinal function as assessed by electroretinography (ERG) was normal in PARP1 KO animals. We then used retinal explant cultures derived from wt, rd1, and PARP1 KO animals to test their susceptibility to chemically induced photoreceptor degeneration. Since photoreceptor degeneration in the rd1 retina is triggered by a loss-of-function in phosphodiesterase-6 (PDE6), we used selective PDE6 inhibition to emulate the rd1 situation on non-rd1 genotypes. While wt retina subjected to PDE6 inhibition showed massive photoreceptor degeneration comparable to rd1 retina, in the PARP1 KO situation, cell death was robustly reduced. Together, these findings demonstrate that PARP1 activity is in principle dispensable for normal retinal function, but is of major importance for photoreceptor degeneration under pathological conditions. Moreover, our results suggest that PARP dependent cell death or PARthanatos may play a major role in retinal degeneration and highlight the possibility to use specific PARP inhibitors for the treatment of RP.

Topics: Animals; Apoptosis; Blotting, Western; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Electroretinography; Female; Humans; In Situ Nick-End Labeling; Male; Mice; Mice, 129 Strain; Mice, Inbred C3H; Mice, Knockout; Phosphodiesterase Inhibitors; Photoreceptor Cells, Vertebrate; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Purinones; Retina; Retinal Degeneration; Retinitis Pigmentosa; Tomography, Optical Coherence

Accumulation of free opsin by mutations in rhodopsin or insufficiencies in the visual cycle can lead to retinal degeneration. Free opsin activates phototransduction; however, the link between constitutive activation and retinal degeneration is unclear. In this study, the photoresponses of Xenopus rods rendered constitutively active by vitamin A deprivation were examined. Unlike their mammalian counterparts, Xenopus rods do not degenerate. Contrasting phototransduction in vitamin A-deprived Xenopus rods with phototransduction in constitutively active mammalian rods may provide new understanding of the mechanisms that lead to retinal degeneration.. The photocurrents of Xenopus tadpole rods were measured with suction electrode recordings, and guanylate cyclase activity was measured with the IBMX (3-isobutyl-1-methylxanthine) jump technique. The amount of rhodopsin in rods was determined by microspectrophotometry.. The vitamin A-deprived rod outer segments were 60% to 70% the length and diameter of the rods in age-matched animals. Approximately 90% of its opsin content was in the free or unbound form. Analogous to bleaching adaptation, the photoresponses were desensitized (10- to 20-fold) and faster. Unlike bleaching adaptation, the vitamin A-deprived rods maintained near normal saturating (dark) current densities by developing abnormally high rates of cGMP synthesis. Their rate of cGMP synthesis in the dark (15 seconds(-1)) was twofold greater than the maximum levels attainable by control rods ( approximately 7 seconds(-1)).. Preserving circulating current density and response range appears to be an important goal for rod homeostasis. However, the compensatory changes associated with vitamin A deprivation in Xenopus rods come at the high metabolic cost of a 15-fold increase in basal ATP consumption.

Topics: Animals; Calbindins; Cyclic GMP; Dark Adaptation; Electrophysiology; Fluorescent Antibody Technique, Indirect; Guanylate Cyclase; Hydrolysis; Light; Microspectrophotometry; Photic Stimulation; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; S100 Calcium Binding Protein G; Vision, Ocular; Vitamin A Deficiency; Xenopus laevis

Mice of the FVB/N strain are severely visual impaired as a result of tyrosinase gene defects, leading to a deficiency of the key enzyme for melanin synthesis in skin and eye and of cyclic guanosine monophosphate phosphodiesterase gene defects, which results in albinism (Tyr(c/c)) and retinal degeneration (Pde6b(rd1/rd1)), respectively. Nevertheless, FVB/N mice are commonly used for the generation of transgenic animals because of their large, strong pronuclei and high breeding performance. However, due to visual impairment of the FVB/N animals, the resulting transgenic animals cannot be used in tests that depend on vision, including tests of cognitive behavior. Therefore, we have bred a sighted version of the FVB/N strain by an outcross between FVB/N and 129P2/OlaHsd, followed by repeated backcrosses to FVB/N mice while selecting against albinism and homozygosity of the retinal degeneration mutation. After 11 generations of backcrossing, sighted animals were intercrossed to generate the congenic FVB.129P2-Pde6b(+) Tyr(c-ch)/Ant strain, which is pigmented (Tyr(c-ch)/(c-ch)) and devoid of the genetic predisposition to retinal degeneration. The accurate visual abilities of the FVB.129P2-Pde6b(+) Tyr(c-ch)/Ant mice, for which we propose the name FVBS/Ant, demonstrated a clear visual evoked potential in the presence of normal eye histology and improved performance in the Morris water maze test.

Topics: Albinism; Animals; Behavioral Research; Crosses, Genetic; Cyclic GMP; Evoked Potentials, Visual; Exploratory Behavior; Eye; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Mutant Strains; Mice, Transgenic; Models, Animal; Monophenol Monooxygenase; Pigmentation; Retinal Degeneration; Species Specificity

Sustained elevation in cGMP and a concomitant increase in intracellular Ca(2+) levels in the rd1 photoreceptors are followed by a rapid loss of photoreceptors. In a murine-derived photoreceptor cell line, 661W, treated with the phosphodiesterase inhibitor IBMX or the cyclic GMP-gated channel agonist 8-bromo-cGMP, it was previously found that the induced cell death was mediated by calpain and caspase-3. Because oxidative stress is a common product of ionic imbalance or elevated Ca(2+), we tested the role of oxidative stress in cGMP-induced photoreceptor cell death. In the rd1 mouse retina, oxidative stress was found to precede calpain and caspase-3 activation. In 661W cells, the increase in intracellular cGMP and Ca(2+) resulted in the generation of reactive oxygen species (ROS), the activation of oxidative stress enzymes, and the activation of calpain, followed by apoptosis mediated by the effector caspase-3. All these events, including calpain activation, were ameliorated by docosahexanoic acid (DHA). The cell-permeable inhibitor of calpain, SJA6017, while inhibiting cell death, had no effect on the generation of oxidative stress. These results establish a central role for oxidative stress in cGMP-induced cell death and suggest a ROS-mediated sequential activation of signal transduction events, which provide targets for future treatment strategies.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Apoptosis; Calcium; Calpain; Caspase 3; Catalase; Cell Line; Cell Survival; Cyclic GMP; Cytochromes c; Docosahexaenoic Acids; Glutathione Peroxidase; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Oxidative Stress; Phosphodiesterase Inhibitors; Photoreceptor Cells, Vertebrate; Reactive Oxygen Species; Retinal Degeneration

Previous experiments indicate that congenital human retinal degeneration caused by genetic mutations that change the Ca(2+) sensitivity of retinal guanylyl cyclase (retGC) can result from an increase in concentration of free intracellular cGMP and Ca(2+) in the photoreceptors. To rescue degeneration in transgenic mouse models having either the Y99C or E155G mutations of the retGC modulator guanylyl cyclase-activating protein 1 (GCAP-1), which produce elevated cGMP synthesis in the dark, we used the G90D rhodopsin mutation, which produces constitutive stimulation of cGMP hydrolysis. The effects of the G90D transgene were evaluated by measuring retGC activity biochemically, by recording single rod and electroretinogram (ERG) responses, by intracellular free Ca(2+) measurement, and by retinal morphological analysis. Although the G90D rhodopsin did not alter the abnormal Ca(2+) sensitivity of retGC in the double-mutant animals, the intracellular free cGMP and Ca(2+) concentrations returned close to normal levels, consistent with constitutive activation of the phosphodiesterase PDE6 cascade in darkness. G90D decreased the light sensitivity of rods but spared them from severe retinal degeneration in Y99C and E155G GCAP-1 mice. More than half of the photoreceptors remained alive, appeared morphologically normal, and produced electrical responses, at the time when their siblings lacking the G90D rhodopsin transgene lost the entire retinal outer nuclear layer and no longer responded to illumination. These experiments indicate that mutations that lead to increases in cGMP and Ca(2+) can trigger photoreceptor degeneration but that constitutive activation of the transduction cascade in these animals can greatly enhance cell survival.

Topics: Adaptation, Ocular; Animals; Aspartic Acid; Calcium; Cyclic GMP; Cysteine; Dark Adaptation; Disease Models, Animal; Electroretinography; Gene Expression Regulation; Glycine; Guanylate Cyclase-Activating Proteins; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Physical Stimulation; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Tyrosine

The effects of various light-induced stresses on the retina were examined in the retinal degenerative rat model.. Retinal morphology and electroretinograms (ERGs) were analyzed after application of light-induced stress of several intensities (650, 1300, 2500, or 5000 lux). For evaluation of rhodopsin (Rho) function, the kinetics of Rho regeneration and dephosphorylation were studied by spectrophotometric analysis and immunofluorescence labeling with antibodies specifically directed toward the phosphorylated residues (334)Ser and (338)Ser in the C terminus of Rho. Retinal cGMP concentration was determined by ELISA. Expression levels of neurotrophic factors (FGF2, brain-derived neurotrophic factor [BDNF], platelet-derived growth factor [PDGF], and ciliary neurotrophic factor [CNTF]) were evaluated quantitatively by RT-PCR.. Light intensity-dependent deterioration of ERG responses and thinning of the retinal outer nuclear layer were observed in wild-type and Royal College of Surgeons (RCS) rat retinas. Under dark adaptation after light-induced stress, the kinetics of Rho regeneration were not different between wild-type and RCS rat retinas. Rho dephosphorylation at (334)Ser and (338)Ser was extremely delayed in RCS rat retinas compared with wild-type without light-induced stress, but Rho dephosphorylation at those sites became slower in both RCS and wild-type rat retinas. In terms of expression of neurotrophic factors, almost no significant changes were observed between the animals after light-induced stress.. The present study indicates that light-induced stress causes intensity-dependent deterioration in retinal function and morphology in wild-type and RCS rat retinas. Disruption of the phototransduction cascade resulting from slower kinetics of Rho dephosphorylation appears to be involved in retinal degeneration.

Topics: Animals; Cyclic GMP; Dark Adaptation; Electroretinography; Enzyme-Linked Immunosorbent Assay; Light; Microscopy, Fluorescence; Nerve Growth Factors; Phosphorylation; Radiation Injuries, Experimental; Rats; Rats, Inbred BN; Rats, Mutant Strains; Rats, Sprague-Dawley; Regeneration; Retina; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger

Achromatopsia 2, an inherited retinal disorder resulting in attenuation or loss of cone function, is caused by mutations in the alpha subunit of the cone cyclic nucleotide-gated (CNG) channel gene CNGA3. Examination of mutations that cluster in the first transmembrane segment of the protein may provide insight into its role in CNG channel structure, function, biogenesis, and pathophysiology.. The human CNGA3 gene was tagged at the C terminus with green fluorescent protein. Four mutations, Y181C, N182Y, L186F, and C191Y, were expressed in human embryonic kidney cells. Protein expression was evaluated with immunoblot analysis and cellular localization was determined by immunocytochemistry. Channel function was evaluated by patch-clamp electrophysiology.. All the mutations result in loss of channel function, as determined by the failure of cGMP to activate wild-type currents in excised patches. Full-length mutant proteins were synthesized but retained in the endoplasmic reticulum. Glycerol treatment did not rescue channel function nor did coexpression with CNGB3, a subunit of native hetero-tetrameric cone channels. A control mutant, C191S, exhibited cGMP current activation with significantly reduced cooperativity, suggesting that mutations in the first transmembrane domain alter in inter- or intrasubunit communication.. The results implicate the first transmembrane segment in both maturation and function of CNG channels. The defects are not reversed with glycerol, a chemical chaperone that rescues channel function in some channelopathies. Molecular analysis of achromatopsia 2 mutations may be useful in evaluating potential therapeutic approaches for treatment of this channelopathy.

Topics: Amino Acid Sequence; Color Vision Defects; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Eye Proteins; Fluorescent Antibody Technique, Indirect; Green Fluorescent Proteins; Humans; Immunoblotting; Ion Channels; Kidney; Molecular Sequence Data; Mutation; Patch-Clamp Techniques; Recombinant Fusion Proteins; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Transfection

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

In the rd/rd mouse, photoreceptor degeneration is due to a mutation of the rod-specific enzyme cGMP phosphodiesterase, resulting in permanently opened cGMP-gated cation channels in the rod outer segment membrane that allow Na(+) and Ca(2+) ions to enter the cell, resulting in possibly toxic levels of Ca(2+). To identify pathways involved in cell death of the rd/rd rods, we evaluated gene expression in the rd/rd and wild type (wt) mouse retina (U74A oligonucleotide arrays (Affymetrix)) over the known time course of photoreceptor degeneration. 181 genes passed the selection criteria (low standard deviation and high correlation between replicates), falling into six clusters. For any given pair of genes, an expression profile correlation distance and a semantic distance (one for each class of gene ontology terms) were established using newly designed software. Gene expression in rd/rd started to deviate from wt by postnatal day 10. The reduction in photoreceptor-specific genes followed the known time course of photoreceptor degeneration. Likewise the increase in transcription factors and apoptosis- and neuroinflammation-specific genes followed the kinetics of the rise in intracellular cGMP in the rod photoreceptors. In addition, genes coding for calcium-binding proteins and those implicated in tissue and vessel remodeling were increased. These results suggest that photoreceptor degeneration in the rd/rd mouse is a process starting with Ca(2+) toxicity followed by secondary insults involving multidestructive pathways such as apoptosis and neuroinflammation, presumably boosting morphological changes. All of these components need to be addressed if rods are to be successfully protected.

Topics: Animals; Calcium; Cell Death; Cluster Analysis; Cyclic GMP; Gene Expression Profiling; Kinetics; Mice; Mice, Knockout; Photoreceptor Cells; Retinal Degeneration; Signal Transduction

The human cone photoreceptor cyclic nucleotide-gated (CNG) channel comprises alpha- and beta-subunits, which are respectively encoded by hCNGA3 and hCNGB3. The purpose was to examine the functional role of hCNGB3 in modulation of human cone CNG channels and to characterize functional consequences of rod monochromacy-associated mutations in hCNGB3 (S435F and D633G).. Macroscopic patch currents were recorded from human embryonic kidney (HEK) 293 cells expressing homomeric (hCNGA3 and hCNGB3) and heteromeric (hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3-D633G) channels using inside-out patch-clamp technique.. Both hCNGA3 homomeric and hCNGA3/hCNGB3 heteromeric channels were activated by cGMP, with half-maximally activating concentration (K(1/2)) of 11.1 +/- 1.0 and 26.2 +/- 1.9 micro M, respectively. The hCNGA3 channels appeared to be more sensitive to inhibition by extracellular Ca(2+) compared with hCNGA3/hCNGB3 channels, when assessed by the degree of outward rectification. Coexpression of either of rod monochromacy-associated mutants of hCNGB3 with hCNGA3 significantly reduced K(1/2) value for cGMP but little affected the sensitivity to extracellular Ca(2+), compared with wild-type heteromeric channels. The selectivity of hCNGA3, hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3-D633G channels for monovalent cations were largely similar. Immunoprecipitation experiments showed association of hCNGA3 subunit with both of wild-type and mutant hCNGB3 subunits.. The hCNGB3 plays an important modulatory role in the function of human cone CNG channels with respect to cGMP and extracellular Ca(2+) sensitivities. The rod monochromacy-associated S435F and D633G mutations in hCNGB3 evokes a significant increase in the apparent affinity for cGMP, which should alter cone function and thereby contribute at least partly to pathogenesis of the disease.

Topics: Calcium; Cell Culture Techniques; Cloning, Molecular; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Gene Expression; Humans; Ion Channels; Kidney; Membrane Potentials; Mutation; Patch-Clamp Techniques; Precipitin Tests; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Transfection

Cyclic GMP (cGMP) has been implicated in the modulation of long-term potentiation (LTP) and depression (LTD) in the hippocampus. Transcripts for subunits of several types of cGMP specific phosphodiesterase are found in the mammalian brain but their relative role in hippocampal function is unclear. The retinal degeneration (rd) mutation in the gene encoding the PDE6B subunit causes a loss of function in PDE6 enzyme and in adult mice homozygous to the mutation it causes blindness. We have used this natural mutation, and the cGMP phosphodiesterase inhibitor zaprinast, in wild-type and rd/rd mouse littermates to investigate whether PDE5 and/or PDE6 regulates excitatory synaptic transmission in the hippocampus. Mice were genotyped using two independent PCR methods. Glutamate-mediated synaptic transmission in the CA1 region or dentate gyrus was unaffected in hippocampal brain slices from mice carrying the rd mutation. Similarly the facilitation of synaptic events by paired-pulse stimuli, and LTP induced by a theta-burst (10 bursts of four events at 100 Hz with a 200-ms inter-burst interval) were normal in rd/rd mice. Inhibition of cGMP-specific PDE activity by zaprinast (10 microM, an inhibitor of PDE5 and PDE6) induced a slowly developing and sustained depression of field synaptic potentials that was quantitatively similar in both wild-type and rd/rd mice. Thus in the CA1 region synaptic plasticity is likely to be regulated by the PDE5 rather than the PDE6 isoform.

Topics: Animals; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Hippocampus; Long-Term Potentiation; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mutation; Neuronal Plasticity; Phosphoric Diester Hydrolases; Rats; Retinal Degeneration; Synapses

To determine whether alterations in rod cGMP-gated channel function mediate retinal degeneration, a transgenic approach in which the alpha subunit of the rod cGMP-gated channel is reduced by the expression of an antisense RNA was used.. A 890-bp fragment of the 5' mouse rod cGMP-gated channel cDNA was cloned in the antisense orientation under the control of the strong bacterial cytomegalovirus promoter. This antisense construct was used to generate transgenic mice in which the expression of the antisense transgene was measured by reverse transcription-polymerase chain reaction. Histologic, immunocytochemical, and TdT-dUTP terminal nick-end labeling (TUNEL) analyses were performed on control and transgenic retina sections to determine the effects of antisense expression on specific cell types.. The antisense RNA was able to inhibit the translation of the rod channel protein in an in vitro assay. Three transgenic mouse lines expressing the antisense construct were obtained. Molecular analyses showed that the antisense is expressed in the eye of each transgenic mouse line, where it reduces rod cGMP-gated channel RNA expression. Histologic and immunocytochemical data showed that transgenic retinas have a reduced number of photoreceptors and bipolar cells. TUNEL staining confirmed that photoreceptor and bipolar cells degenerate along an apoptotic pathway.. Impairment of rod cGMP-gated channel alpha subunit expression leads to photoreceptor and bipolar cell degeneration. These transgenic mice are the first model of retinal degeneration caused by an alteration in the expression of the rod cGMP-gated channel. This model system can be used to test therapies designed to slow or stalled retinal degenerations.

Topics: Animals; Apoptosis; Blotting, Northern; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Eye Proteins; Gene Expression; In Situ Nick-End Labeling; Interneurons; Ion Channels; Mice; Mice, Transgenic; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Antisense

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

Photoreceptor membrane guanylate cyclases (RetGC) are regulated by calcium-binding proteins, GCAP-1 and GCAP-2. At Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, guanylate cyclase-activating protein (GCAPs) activate RetGC, and at free Ca2+ concentrations above 500 nM, characteristic of dark-adapted photoreceptors, GCAPs inhibit RetGC. A mutation, Y99C, in human GCAP-1 was recently found to be linked to autosomal dominant cone dystrophy in a British family (Payne, A. M., Downes, S. M., Bessant, D. A. R., Taylor, R., Holder, G. E., Warren, M. J., Bird, A. C., and Bhattachraya, S. S. (1998) Hum. Mol. Genet. 7, 273-277). We produced recombinant Y99C GCAP-1 mutant and tested its ability to activate RetGC in vitro at various free Ca2+ concentrations. The Y99C mutation does not decrease the ability of GCAP-1 to activate RetGC. However, RetGC stimulated by the Y99C GCAP-1 remains active even at Ca2+ concentration above 1 microM. Hence, the cyclase becomes constitutively active within the whole physiologically relevant range of free Ca2+ concentrations. We have also found that the Y99C GCAP-1 can activate RetGC even in the presence of Ca2+-loaded nonmutant GCAPs. This is consistent with the fact that cone degeneration was dominant in human patients who carried such mutation (Payne, A. M., Downes, S. M., Bessant, D. A. R. , Taylor, R., Holder, G. E., Warren, M. J., Bird, A. C., and Bhattachraya, S. S. (1998) Hum. Mol. Genet. 7, 273-277). A similar mutation, Y104C, in GCAP-2 results in a different phenotype. This mutation apparently does not affect Ca2+ sensitivity of GCAP-2. Instead, the Y104C GCAP-2 stimulates RetGC less efficiently than the wild-type GCAP-2. Our data indicate that cone degeneration associated with the Y99C mutation in GCAP-1 can be a result of constitutive activation of cGMP synthesis.

Topics: Amino Acid Sequence; Calcium; Calcium-Binding Proteins; Cyclic GMP; DNA Primers; Enzyme Activation; Guanylate Cyclase; Guanylate Cyclase-Activating Proteins; Humans; Molecular Sequence Data; Photoreceptor Cells; Recombinant Proteins; Retinal Cone Photoreceptor Cells; Retinal Degeneration

The nucleotide sequence of the canine rod photoreceptor cGMP-gated cation channel alpha-subunit (cCNCG1) cDNA has been determined. The open reading frame consists of 2073 nucleotides, which encode a putative protein of 691 amino acids. In addition, we have established the exon/intron boundaries of the cCNCG1 gene and determined the complete sequence of six introns of a total of eight. The exon/intron organization (location and length of exons and introns) of the cCNCG1 gene is very similar to that of the human rod photoreceptor cGMP-gated cation channel alpha-subunit gene. We used single-strand conformation polymorphism analysis to search for potential pathogenic sequence changes in the cCNCG1 gene in a Swedish Briard and Briard-Beagle dog kindred, in which an autosomal recessive retinal dystrophy is segregating, a disease which shows phenotypic similarities to retinitis pigmentosa, a heterogeneous group of hereditary and progressive retinal degeneration in human. In intron 3, we found several DNA polymorphisms, which do not cosegregate with the affected status of the dogs, thus excluding cCNCG1 as a candidate gene for the retinal dystrophy in this strain of Swedish Briards.

Topics: Amino Acid Sequence; Animals; Cattle; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; DNA, Complementary; Dog Diseases; Dogs; Exons; Humans; Inbreeding; Introns; Ion Channels; Mice; Molecular Sequence Data; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Rats; Retinal Degeneration; Rod Cell Outer Segment; Sequence Homology, Amino Acid; Sweden

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.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Base Sequence; Chimera; Crosses, Genetic; Cyclic GMP; Electroretinography; Enzyme Activation; Female; Gene Targeting; Humans; Light; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Phenotype; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa

Topics: Animals; Cyclic GMP; Humans; Ion Channel Gating; Ion Channels; Mice; Mutation; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Unusual rod electroretinogram (ERG) intensity-response functions were recorded from three female patients with retinal degeneration who had visual acuities of 20/200, retinal arteriolar narrowing, and diffuse granularity of the retinal pigment epithelium. All three patients had rod b-waves that were profoundly subnormal in amplitude and markedly delayed in implicit time to bright stimuli. Rod a-wave slopes were reduced 50% below normal, indicating photoreceptor involvement. These unusual rod ERG intensity-response functions are similar to those previously reported for the isolated cat eye with elevated retinal cyclic guanosine monophosphate (cGMP) after perfusion with isobutylmethylxanthine. This finding supports the idea that these three patients may have an elevation of retinal cGMP. Their rod ERG intensity-response functions are contrasted with those recorded from some patients with retinitis pigmentosa.

Topics: 1-Methyl-3-isobutylxanthine; Adolescent; Adult; Animals; Cats; Child; Cyclic GMP; Electroretinography; Female; Humans; Middle Aged; Photoreceptor Cells; Retina; Retinal Degeneration; Retinitis Pigmentosa; Visual Acuity

Mice homozygous for the rd mutation display hereditary retinal degeneration and the classic rd lines serve as a model for human retinitis pigmentosa. In affected animals the retinal rod photoreceptor cells begin degenerating at about postnatal day 8, and by four weeks no photoreceptors are left. Degeneration is preceded by accumulation of cyclic GMP in the retina and is correlated with deficient activity of the rod photoreceptor cGMP-phosphodiesterase. We have recently isolated a candidate complementary DNA for the rd gene from a mouse retinal library and completed the characterization of cDNAs encoding all subunits of bovine photoreceptor phosphodiesterase. The candidate cDNA shows strong homology with a cDNA encoding the bovine phosphodiesterase beta subunit. Here we present evidence that the candidate cDNA is the murine homologue of bovine phosphodiesterase beta cDNA. We conclude that the mouse rd locus encodes the rod photoreceptor cGMP-phosphodiesterase beta subunit.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Amino Acid Sequence; Animals; Base Sequence; Chromosome Mapping; Cloning, Molecular; Cyclic GMP; DNA; DNA Probes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Mutant Strains; Molecular Sequence Data; Nucleic Acid Hybridization; Photoreceptor Cells; Polymorphism, Restriction Fragment Length; Retina; Retinal Degeneration

Topics: Animals; Cats; Cyclic GMP; Phosphorylation; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

Cross-breeding experiments were utilized to study the genetics of three autosomal recessive, early onset retinal degenerations in dogs. Irish setters affected with rod-cone dysplasia type 1 (rcd1) were bred to Norwegian elkhounds affected with early retinal degeneration (erd). All offspring (15 pups-two litters) surviving to diagnostic age were phenotypically normal, as assessed by electroretinography, retinal morphology and assay of retinal cyclic nucleotide content. One phenotypically normal female Irish setter-Norwegian elkhound crossbred dog (the progeny of the above breeding and thus heterozygous at both the rcd1 and the erd locus) was bred to a collie dog affected with rod-cone dysplasia type 2 (rcd2). All 11 pups from this breeding also proved phenotypically normal by the above methods. These results establish that the genes rcd1, rcd2 and erd are non-allelic. Biochemical data are also presented that establish that erd, unlike rcd1 and rcd2, is not associated with abnormal metabolism of retinal cyclic guanosine monophosphate (cyclic GMP).

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Choroid; Cyclic AMP; Cyclic GMP; Dogs; Female; Genes, Recessive; Genotype; Male; Pedigree; Pigment Epithelium of Eye; Retina; Retinal Degeneration

Electroretinographic and cyclic nucleotide metabolism studies have established that low-level lead exposure during early postnatal development results in long-term selective rod deficits. To determine whether there was a corresponding selective rod photoreceptor cell degeneration we examined retinas of adult rats exposed to low-level lead during development using light and electron microscopy. In all retinal regions, a rod but not cone cell degeneration was observed. Overall, 20% of the rod cells were lost. Moreover, two specific regional differences were found. Degeneration was much greater in the inferior (-25%) than superior (-15%) retina and greater in the posterior (-22%) than peripheral (-17%) retina. The latter pattern indicates a central-peripheral gradient of degeneration. Total retinal thickness decreased 15-20%, which reflects cell loss in the outer and inner nuclear layers. Ultrastructurally, the most obvious lead-induced alterations were swollen and disorganized rod outer segments and large accumulations of beta-glycogen particles in rod photoreceptor mitochondria. Glycogen accumulations were heaviest in rod inner segment mitochondria followed by rod axon and synaptic terminal mitochondria. Possible cellular mechanisms of action responsible for these lead-induced retinal alterations include an inhibition of retinal cyclic GMP phosphodiesterase and the resultant elevation of cyclic GMP, an inhibition of intermediary metabolism, and/or an alteration in calcium metabolism. In addition, the thinning of the inner nuclear layers could be due to transneuronal degeneration. As noted in our preceding paper, the first possibility has been demonstrated in rats similarly exposed to lead. These quantitative histological results, in combination with the ERG and biochemical results in the preceding paper, demonstrate that low-level lead exposure during early postnatal development produces long-term selective rod functional deficits and degeneration.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Female; Glycogen; Lead; Microscopy, Electron; Mitochondria; Photoreceptor Cells; Rats; Retinal Degeneration; Rod Cell Outer Segment

Retinal degeneration in rd mice is manifested during the most rapid period of postnatal photoreceptor differentiation and is hypothesized to be caused by a lesion in cGMP metabolism. We have studied the sequence of developmental expression of three proteins involved in the cGMP cascade and the mRNAs from which they are translated, in rd and control mouse retinas. Slot blot analysis of retinal RNAs indicates that the mRNAs coding for opsin, the alpha, beta and gamma subunits of G-protein and 48-kDa protein each has the same time for onset of expression in normal and diseased retinas. G beta and 48-kDa protein mRNAs are already detectable at birth, opsin mRNA appears by postnatal day 5 (P5), G gamma mRNA at P6 and G alpha mRNA by P8. The levels of all these mRNAs decrease in the diseased retinas after P11-P12, correlating with the reduction in photoreceptor cell number that characterizes the rd disease. Immunocytochemistry indicates that the 48-kDa protein is present at birth, G gamma and opsin are detectable at P4 and G alpha at P7. After P7, opsin and G-protein immunoreactivity are localized throughout the photoreceptor cell in the rd retinas but they are found only in the outer segment in control retinas. The 48-kDa protein immunoreactivity, which is observed in the whole photoreceptor layer both in rd and control retinas throughout development, is the only one of all immunoreactivities analysed that remains at 2 months of age in the rd retina and is probably localized in cones. However, at 6 months of age, 48-kDa protein immunoreactive cells are no longer present in the rd retina. We have also investigated whether there is a daily rhythm for the levels of mRNA present at different times during the light/dark periods in developing rd/rd and rd/+ retinas and in adult normal (+/+) retinas. We find that the levels of each mRNA analysed appear to cycle in the +/+ adult retina, with the greatest amount of opsin and the three subunits of G-protein mRNAs occurring just before light onset and the greatest amount of 48-kDa protein mRNA occurring just before lights off. Cycling in the developing diseased or control retinas (P0-P12) is not observed and may be masked by the pronounced cell growth that occurs during this period.

Topics: Aging; Animals; Circadian Rhythm; Cyclic GMP; Eye Proteins; GTP-Binding Protein alpha Subunits; GTP-Binding Proteins; Heterotrimeric GTP-Binding Proteins; Immunohistochemistry; Light; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration; RNA, Messenger; Rod Opsins; Transducin

Light-adapted retinas from Irish setters affected with inherited rod-cone dysplasia accumulate high concentrations of cyclic GMP in the outer plexiform layer (OPL). A 29-fold difference in OPL cGMP levels between affected and normal occurs at 19-20 days. However, the highest concentration, 550 mumol kg-1 (dry) wt, is reached at about 4 weeks of age, at which time OPL cyclic GMP is 20-fold higher than cGMP in the OPL of normal control retinas. Levels remain high in affected OPL until about 7 weeks of age; after this, the cyclic GMP level falls and the peak shifts to the outer nuclear layer (ONL). In the normal retina on the other hand, the highest cyclic GMP levels are in the outer segments (OS). The result is that cyclic GMP is highest at opposite ends of the photoreceptor neuron in normal and affected retinas during the first 7 weeks of development. These differing distributions are established as early as postnatal day 10, before signs of degeneration become evident, as determined by either ERG or morphological examination. Moreover, a 38-fold rise in cyclic GMP occurs in the ONL of affected retinas between 1 and 3 weeks of age significantly before the degeneration of nuclei begins at 4 to 5 weeks. The early differences in cyclic GMP distribution are probably not due to differences in guanylate cyclase activity since enzyme levels in normal and affected photoreceptor cells are virtually identical until about 4 weeks of age. Since cGMP has been observed to reach high levels in normal dark-adapted rabbit and frog OPL, the extraordinary OPL levels seen in affected setters may indicate the importance of cGMP in both normal synaptic function and in the disease process.

Topics: Aging; Animals; Animals, Newborn; Cyclic GMP; Dog Diseases; Dogs; Guanylate Cyclase; Photoreceptor Cells; Retina; Retinal Degeneration

In the retina of the mouse heterozygous for retinal degeneration (+ rd) cGMP levels are reported to be considerably reduced from normal. The sensitivity and timing of the a- and b-wave components of the electroretinogram of anaesthetized, dark-adapted, heterozygous (+ rd) mice were measured and compared with those of normals (+ +). The (+ rd) mouse was found to be 0.43 log units more sensitive to flashes of green light than the normal for both a- and b-waves; the Vmax amplitude of the b-wave was larger; and variations in time-to-peak were of small magnitude except at low a-wave intensities, where the + rd responses were slowed compared with those of + +. Reduced phosphodiesterase activity has been suggested previously as a mechanism for altered cGMP levels in the rd mouse. The findings reported are interpreted in terms of reduced rates of cGMP metabolism.

Topics: Animals; Cyclic GMP; Electroretinography; Heterozygote; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration

Topics: Animals; Cyclic GMP; Disease Models, Animal; Dog Diseases; Dogs; Mice; Mice, Mutant Strains; Mutation; Nucleotides, Cyclic; Phosphoric Diester Hydrolases; Retina; Retinal Degeneration; Rodent Diseases

A lesion in cGMP metabolism has been hypothesized to cause retinal degeneration in rd mice. Available cloned cDNAs coding for proteins involved in the cGMP cascade have been used to compare the corresponding retinal RNAs in the degenerative (rd/rd) mouse at 8-11 days with those in the 8-11-day-old morphologically normal (rd/+) and adult normal (+/+) mice. Northern analysis of these RNAs hybridized to the specific 32P-labeled cDNA probes for G-protein, 48,000 MW protein and opsin, indicates in each case, that the corresponding transcripts are made in the rd/rd mouse retina and that there are no overt differences in their size compared to the transcripts hybridized in the rd/+ or +/+ mouse retinas. Although a defect in the phosphorylation of opsin has been described in rd mice, no difference was found in the transcripts hybridized by a cDNA probe corresponding to the region of the opsin molecule on which phosphorylation occurs. We do find, however, that labeled bovine-derived opsin cDNA recognizes five different RNA size classes in mouse and bovine retinas. Control experiments were performed to confirm that the RNA hybridized by opsin cDNA was not due to non-specific hybridization to unrelated RNAs.

Topics: Animals; Antigens; Arrestin; Cyclic GMP; DNA, Recombinant; Electrophoresis, Agar Gel; Eye Proteins; GTP-Binding Protein alpha Subunits; GTP-Binding Proteins; Heterotrimeric GTP-Binding Proteins; Mice; Mice, Mutant Strains; Molecular Weight; Nucleic Acid Hybridization; Retina; Retinal Degeneration; Retinal Pigments; RNA, Messenger; Rod Opsins; Transcription, Genetic; Transducin

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.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Female; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Phosphorylation; Photoreceptor Cells; Rabbits; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Rod Cell Outer Segment

We have examined cyclic GMP concentrations, guanylate cyclase activities, and cyclic GMP phosphodiesterase (PDE) activities in developing retinas of congenic mice with different allelic combinations at the retinal degeneration (rd) and retinal degeneration slow (rds) loci. Although guanylate cyclase activities were found to be uniformly low in the mutant retinas, striking differences in PDE activity and cyclic GMP levels were observed in retinas of the various genotypes. Homozygous rds mice, which lack receptor outer segments, showed reduced retinal PDE activity and cyclic GMP concentration in comparison to normal animals. In heterozygous rds/+ mice with abnormal outer segments, the levels were intermediate. In retinas of homozygous rd mice, PDE activity was lower than in rds retinas and cyclic GMP levels were much higher. In mice homozygous for both rd and rds genes, retinal PDE activities were even lower than in single homozygous rd mice; the cyclic GMP level reached the same high value as in the rd animals, persisted for a longer time at this high level, and did not correlate with the rate of photoreceptor cell loss. Thus, a marked variation in PDE activity appears to be the major manifestation of abnormal outer segment differentiation and eventual degeneration of photoreceptor cells in these neurological mutants. An increased cyclic GMP level seems to be an essential corollary in the expression of the rd gene even in the absence of outer segments, but it appears unlikely that an abnormally high nucleotide level in itself causes photoreceptor cell death.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic AMP; Cyclic GMP; Mice; Mice, Neurologic Mutants; Photoreceptor Cells; Retina; Retinal Degeneration

An unusual form of retinal degeneration is reported in 15-year-old girl and 11-year-old girl with different pedigrees, which resembles the cases reported by Gouras and associates (1983). The subjective symptoms in these patients included decreased visual acuity, photophobia, anomalous color vision and night blindness. Electroretinograms (ERGs) in these two patients were identical in substance and revealed drastic alterations in both photopic and scotopic functions. The stimulus versus intensity response curve in a single-flash ERG showed an unusual form. This peculiar supernormal response was elicited by bright stimuli although the stimulus threshold was extremely elevated.

Topics: Adolescent; Child; Cyclic GMP; Dark Adaptation; Electrooculography; Electroretinography; Female; Humans; Retinal Degeneration

A deficiency in light-dependent opsin phosphorylation and a slight reduction in opsin synthesis were observed during photoreceptor cell development (22-26 days) preceding photoreceptor cell loss in Irish setters with rod-cone dysplasia. In addition to opsin, two other phosphoprotein bands were found associated with the photoreceptor cell layer; synthesis and phosphorylation of one of these (band 3; 44-48 Kd) appeared reduced, while synthesis and phosphorylation of the other (band 1; 29-31 Kd) was within the normal range in 25-day-old affected setters. The deficiency in light-dependent opsin phosphorylation in affected setters was not due to a deficiency in opsin kinase, since soluble proteins from affected or normal outer segments catalyzed equally well opsin phosphorylation in partially kinase-depleted outer segment membranes from normal, while both kinase preparations failed to promote light-dependent opsin phosphorylation in those from affected setters. A deficiency in light-dependent opsin phosphorylation was also observed in rd/rd mice at all ages studied. In contrast, in Royal College of Surgeons (RCS) rats, light-dependent opsin phosphorylation was within the normal range prior to photoreceptor loss, and became nondetectable only after 50% or more of the photoreceptors had degenerated.

Topics: Animals; Cyclic GMP; Dogs; Electrophoresis; Eye Proteins; G-Protein-Coupled Receptor Kinase 1; Phosphorylation; Photoreceptor Cells; Protein Kinases; Radioligand Assay; Retinal Degeneration; Rod Opsins

Cyclic GMP metabolism has been investigated in the retinas of mice that are heterozygous for a 'photoreceptor dystrophy' gene and have a lowered concentration of cGMP in their photoreceptor cells. The concentration of rhodopsin, retinal morphology and guanylate cyclase kinetics were normal. Cyclic GMP phosphodiesterase had a lowered affinity for cGMP. In accord with previous observations, chelation of exogenous calcium had no effect on cGMP levels in light-adapted retinas but increased them in dark-adapted tissue. The difference between cGMP concentrations in heterozygous and normal retinas in the dark was then eliminated. It was concluded that a modulator of cGMP phosphodiesterase activity is most likely to be causing the lowered steady-state level of cGMP in heterozygous retinas and that calcium is not involved.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calcium; Cyclic GMP; Dark Adaptation; Guanylate Cyclase; Heterozygote; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration; Rhodopsin

The phosphodiesterase from the visual cells of rd mice and affected Irish setter dogs has been analyzed, using biochemical, biophysical, and immunological techniques. The authors' findings demonstrate that the mechanisms that cause a deficiency in phosphodiesterase activity in rd mice and Irish setter dogs are distinctly different. Apparently, the phosphodiesterase complex is normal in affected Irish setter dogs but is abnormal in rd mice. The criteria used for determining the normalcy of the phosphodiesterase complex were sedimentation characteristics, immuno-cross-reactivity, and histone-activation, which is shown to be a unique characteristic of the visual cell enzyme. According to these criteria, the phosphodiesterase complex in the visual cells of rd mice is either absent or abnormal from the onset of visual cell differentiation until degeneration, because it exhibits no cross-reactivity with antibody to phosphodiesterase; it is not activated by histone; and if present, it exhibits abnormal sedimentation characteristics and perhaps subunit structure. On the other hand, phosphodiesterase from the visual cells of affected Irish setter dogs is normal by the same criteria, because it cross-reacts with antibody against phosphodiesterase; it is activated by histone; and it exhibits normal sedimentation and electrophoretic patterns. It is proposed that depressed levels of phosphodiesterase activity in affected setter photoreceptors are due, perhaps, to a defect in the light-initiated cascade which activates the enzyme normally, in situ.

Topics: Animals; Anura; Brain; Cattle; Cyclic GMP; Dog Diseases; Dogs; Histones; Hydrolysis; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Molecular Weight; Phosphoric Diester Hydrolases; Photoreceptor Cells; Rats; Retina; Retinal Degeneration; Rod Cell Outer Segment; Species Specificity

Retinas obtained from 7-day-old rd mice show less reaction with antirhodopsin antisera than retinas from normal mice of the same age. Likewise, antisera prepared against synthetic peptides, which corresponds to the carboxyl terminus of rhodopsin, also react less with rd retinas from 7-day-old mice. In contrast, Western blots of denatured rhodopsin from rd vs. normal retinas of the same age indicate no change in the total quantity of this protein. These results demonstrate that in the 7-day-old rd mouse retina, rhodopsin is not altered in quantity; rather, it is less accessible to reaction with anti-rhodopsin antisera. Furthermore, these results suggest that the site of altered accessibility is on the carboxyl terminus of rhodopsin.

Topics: Age Factors; Animals; Calmodulin; Cyclic GMP; Immunologic Techniques; Mice; Mice, Mutant Strains; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Rod Cell Outer Segment

An asymptomatic 19-year-old male with choroideremia had diffuse loss of retinal pigment epithelium (RPE) and choroid except for the periphery and macula. Fluorescein angiography of the arteriovenous phase showed absence of retinal pigment epithelium and exaggerated visualization of choroidal vessels in involved areas. The mother was a typical carrier with pigment stippling of the midperipheral retina. Histopathologic examination of affected areas of one eye showed marked degeneration of the outer and midretina with loss of retinal pigment epithelium and Bruch's membrane, absence of choriocapillaris, chorioretinal adhesions and gliosis. Atrophy of inner and mid-choroid was also observed. Pigmented macrophage-like cells had migrated into the outer and midretinal layers. Electron microscopy disclosed macrophage-like cells with trilaminar structures and photoreceptor phagosomes in the RPE and outer retina. Remnants of photoreceptor outer segments were adherent to the plasma membranes of the macrophage-like cells. Biochemical analysis of retinal tissue samples for interphotoreceptor retinoid-binding protein (IRBP) showed marked reduction in the 146K bands in the equator and posterior pole in the patient compared to controls. Cyclic nucleotide content was altered in the retinal equator. Cyclic AMP was several-fold higher in the RPE-choroid complex of the affected eye than in the control.

Topics: Adult; Choroid; Cyclic AMP; Cyclic GMP; Cytosol; Electrophoresis, Polyacrylamide Gel; Fluorescein Angiography; Humans; Male; Microscopy, Electron; Photoreceptor Cells; Pigment Epithelium of Eye; Retina; Retinal Degeneration; Uveal Diseases

Cyclic nucleotide metabolism was examined in the retina and in the retinal pigment epithelium (RPE)-choroid complex of the rds mouse (020/A), a mutant in which discrete photoreceptor outer segment disc structures fail to develop. In retinas of both rds and control (Balb/c) mice, cyclic AMP levels peak at 10-15 days (20-25 pmol mg-1 protein). The level drops to about 10 pmol mg-1 at about one month in normal retinas but remains high in affected retinas. Cyclic GMP levels increase five-fold in Balb/c retinas as ROS develop whereas, in affected retinas, the levels remain constant and low (about 5 pmol mg-1). In RPE-choroid, cyclic nucleotide levels are similar in control and affected mice. Cyclic AMP phosphodiesterase (PDE) activity is somewhat higher in affected than in control retinas; conversely, cyclic GMP-PDE is lower. Both cyclic AMP-PDE and cyclic GMP-PDE activities are different in normal and affected RPE-choroid. Thus, although the rds (020/A) mouse belongs to the early-onset photoreceptor dysplasia group of hereditary retinal degenerations on a morphological basis, it does not exhibit high retinal cyclic GMP levels and thus appears to be distinct from other animals exhibiting early postnatal photoreceptor dysfunction.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Age Factors; Animals; Cyclic AMP; Cyclic GMP; Mice; Mice, Mutant Strains; Mutation; Pigment Epithelium of Eye; Retina; Retinal Degeneration

Incubation of mouse or rabbit whole retina homogenates in the presence of [gamma 32P]-ATP and Mg2+ leads to the phosphorylation of various proteins, as demonstrated using SDS-polyacrylamide gel electrophoresis and autoradiography. The phosphorylation of one protein (ca. approximately equal to 31000 mol. wt) was increased by cyclic AMP in both species, with half-maximal stimulation at 5 x 10(-7)M. Cyclic GMP was also active, but much less potent. Protein phosphorylation patterns were compared in retina homogenates from normal mice (C57BL/6J), from adult C57BL/6J mice homozygous for the retinal degeneration gene (rd/rd) in which rod photoreceptor cells are absent, and from 21-day-old 020/Cpb mice homozygous for the retinal degeneration slow gene (rds/rds) in which only the outer segments of the rod photoreceptors are missing. The 31 K protein was only present in normal and in 21-day-old rds/rds mice. When rabbit retina was microdissected into outer segment, inner segment plus outer nuclear, and inner retina layers, cyclic AMP-stimulated phosphorylation of the 31 K protein was evident only in the inner segment plus outer nuclear layer. These data indicated the presence of a specific, endogenous substrate for a cAMP-dependent protein kinase which is found in the inner portions of rod photoreceptor cells.

Topics: Animals; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Eye Proteins; In Vitro Techniques; Phosphoproteins; Phosphorylation; Rabbits; Retina; Retinal Degeneration; Stimulation, Chemical

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calmodulin; Cyclic AMP; Cyclic GMP; Dog Diseases; Dogs; Histocytochemistry; Microscopy, Electron; Retina; Retinal Degeneration

Topics: Animals; Cyclic AMP; Cyclic GMP; Dog Diseases; Dogs; Genes, Recessive; Photoreceptor Cells; Retina; Retinal Degeneration

Topics: Animals; Animals, Newborn; Cyclic GMP; Dogs; Eye Proteins; Microscopy, Electron; Nucleotides, Cyclic; Photoreceptor Cells; Retina; Retinal Degeneration

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Calcium; Cell Differentiation; Cell Survival; Cyclic GMP; Enzyme Activation; Light; Mice; Mice, Inbred Strains; Retina; Retinal Degeneration

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Adenylyl Cyclases; Animals; Cyclic AMP; Cyclic GMP; Dogs; Dopamine; Mice; Nucleotides, Cyclic; Photoreceptor Cells; Rabbits; Rats; Retina; Retinal Degeneration; Rod Cell Outer Segment; Vertebrates

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic AMP; Cyclic GMP; Histocytochemistry; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Phosphoric Diester Hydrolases; Photoreceptor Cells; Retinal Degeneration

An abnormality in retinal guanosine 3,5-monophosphate (cyclic GMP) metabolism is demonstrated in the inherited rod-cone dysplasis of Irish Setter dogs. Affected visual cells are deficient in cyclic GMP phosphodiesterase activity and have elevated levels of cyclic GMP. The biochemical abnormalities observed in affected retinas of Irish Setters are similar to those in the retinas of mice with inherited retinal degeneration before visual cell degeneration begins. A defect in cyclic GMP metabolism may be characteristic of early-onset degenerative diseases of the retina, possibly including those that affect humans.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cell Differentiation; Chromosome Aberrations; Chromosome Disorders; Cyclic GMP; Dog Diseases; Dogs; Kinetics; Mice; Photoreceptor Cells; Retina; Retinal Degeneration

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.

Topics: Animals; Cyclic GMP; Microscopy, Electron; Phosphodiesterase Inhibitors; Photoreceptor Cells; Retinal Degeneration; Xanthines; Xenopus

Topics: Adenosine Triphosphate; Animals; Cyclic AMP; Cyclic GMP; Enzyme Activation; Guanylate Cyclase; Light; Magnesium; Phosphoric Diester Hydrolases; Photoreceptor Cells; Protein Kinases; Rana catesbeiana; Retina; Retinal Degeneration; Visual Perception

Elevated levels of guanosine 3'-5'-monophosphate (cyclic GMP) are associated with photoreceptor degeneration in the retina of C3H/HeJ mice. This abonormality has been shown to result from a deficiency in the activity of a receptor-specific cyclic GMP phosphodiesterase. The apparent susceptibility of photoreceptor cells to an abnormality in cyclic GMP metabolism suggests that cyclic GMP may play a role in photoreceptor function which is essential to the viability of these specialized cells.

Topics: Animals; Cyclic GMP; Disease Models, Animal; Mice; Mice, Inbred C3H; Phosphoric Diester Hydrolases; Photoreceptor Cells; Retina; Retinal Degeneration

Topics: Age Factors; Animals; Calcium; Cyclic GMP; DNA; Magnesium; Mice; Photoreceptor Cells; Rats; Retina; Retinal Degeneration; Rhodopsin

Topics: Animals; Cyclic AMP; Cyclic GMP; Phosphoric Diester Hydrolases; Photoreceptor Cells; Rats; Rats, Inbred Strains; Retina; Retinal Degeneration; Retinitis Pigmentosa

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Age Factors; Animals; Cell Fractionation; Cyclic AMP; Cyclic GMP; Dialysis; Female; Freeze Drying; Hot Temperature; In Vitro Techniques; Kinetics; Male; Phosphoric Diester Hydrolases; Photoreceptor Cells; Proteins; Rats; Retinal Degeneration

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Animals; Anura; Cattle; Cyclic AMP; Cyclic GMP; Guanosine Triphosphate; Guanylate Cyclase; Mice; Mice, Inbred C3H; Phosphoric Diester Hydrolases; Photoreceptor Cells; Protein Kinases; Rats; Retina; Retinal Degeneration