Retinal degeneration-3 protein attenuates photoreceptor degeneration in transgenic mice expressing dominant mutation of human retinal guanylyl cyclase.

Different forms of photoreceptor degeneration cause blindness. Retinal degeneration-3 protein (RD3) deficiency in photoreceptors leads to recessive congenital blindness. We proposed that aberrant activation of the retinal membrane guanylyl cyclase (RetGC) by its calcium-sensor proteins (guanylyl cyclase-activating protein [GCAP]) causes this retinal degeneration and that RD3 protects photoreceptors by preventing such activation. We here present in vivo evidence that RD3 protects photoreceptors by suppressing activation of both RetGC1 and RetGC2 isozymes. We further suggested that insufficient inhibition of RetGC by RD3 could contribute to some dominant forms of retinal degeneration. The R838S substitution in RetGC1 that causes autosomal-dominant cone-rod dystrophy 6, not only impedes deceleration of RetGC1 activity by Ca2+GCAPs but also elevates this isozyme's resistance to inhibition by RD3. We found that RD3 prolongs the survival of photoreceptors in transgenic mice harboring human R838S RetGC1 (R838S+). Overexpression of GFP-tagged human RD3 did not improve the calcium sensitivity of cGMP production in R838S+ retinas but slowed the progression of retinal blindness and photoreceptor degeneration. Fluorescence of the GFP-tagged RD3 in the retina only partially overlapped with immunofluorescence of RetGC1 or GCAP1, indicating that RD3 separates from the enzyme before the RetGC1:GCAP1 complex is formed in the photoreceptor outer segment. Most importantly, our in vivo results indicate that, in addition to the abnormal Ca2+ sensitivity of R838S RetGC1 in the outer segment, the mutated RetGC1 becomes resistant to inhibition by RD3 in a different cellular compartment(s) and suggest that RD3 overexpression could be utilized to reduce the severity of cone-rod dystrophy 6 pathology.

Retinal degeneration-3 protein promotes photoreceptor survival by suppressing activation of guanylyl cyclase rather than accelerating GMP recycling.

Retinal degeneration-3 protein (RD3) deficiency causes photoreceptor dysfunction and rapid degeneration in the rd3 mouse strain and in human Leber's congenital amaurosis, a congenital retinal dystrophy that results in early vision loss. However, the mechanisms responsible for photoreceptor death remain unclear. Here, we tested two hypothesized biochemical events that may underlie photoreceptor death: (i) the failure to prevent aberrant activation of retinal guanylyl cyclase (RetGC) by calcium-sensor proteins (GCAPs) versus (ii) the reduction of GMP phosphorylation rate, preventing its recycling to GDP/GTP. We found that GMP converts to GDP/GTP in the photoreceptor fraction of the retina ∼24-fold faster in WT mice and ∼400-fold faster in rd3 mice than GTP conversion to cGMP by RetGC. Adding purified RD3 to the retinal extracts inhibited RetGC 4-fold but did not affect GMP phosphorylation in wildtype or rd3 retinas. RD3-deficient photoreceptors rapidly degenerated in rd3 mice that were reared in constant darkness to prevent light-activated GTP consumption via RetGC and phosphodiesterase 6. In contrast, rd3 degeneration was alleviated by deletion of GCAPs. After 2.5 months, only ∼40% of photoreceptors remained in rd3/rd3 retinas. Deletion of GCAP1 or GCAP2 alone preserved 68% and 57% of photoreceptors, respectively, whereas deletion of GCAP1 and GCAP2 together preserved 86%. Taken together, our in vitro and in vivo results support the hypothesis that RD3 prevents photoreceptor death primarily by suppressing activation of RetGC by both GCAP1 and GCAP2 but do not support the hypothesis that RD3 plays a significant role in GMP recycling.

Two clusters of surface-exposed amino acid residues enable high-affinity binding of retinal degeneration-3 (RD3) protein to retinal guanylyl cyclase.

Retinal degeneration-3 (RD3) protein protects photoreceptors from degeneration by preventing retinal guanylyl cyclase (RetGC) activation via calcium-sensing guanylyl cyclase-activating proteins (GCAP), and RD3 truncation causes severe congenital blindness in humans and other animals. The three-dimensional structure of RD3 has recently been established, but the molecular mechanisms of its inhibitory binding to RetGC remain unclear. Here, we report the results of probing 133 surface-exposed residues in RD3 by single substitutions and deletions to identify side chains that are critical for the inhibitory binding of RD3 to RetGC. We tested the effects of these substitutions and deletions in vitro by reconstituting purified RD3 variants with GCAP1-activated human RetGC1. Although the vast majority of the surface-exposed residues tolerated substitutions without loss of RD3's inhibitory activity, substitutions in two distinct narrow clusters located on the opposite sides of the molecule effectively suppressed RD3 binding to the cyclase. The first surface-exposed cluster included residues adjacent to Leu63 in the loop connecting helices 1 and 2. The second cluster surrounded Arg101 on a surface of helix 3. Single substitutions in those two clusters drastically, i.e. up to 245-fold, reduced the IC50 for the cyclase inhibition. Inactivation of the two binding sites completely disabled binding of RD3 to RetGC1 in living HEK293 cells. In contrast, deletion of 49 C-terminal residues did not affect the apparent affinity of RD3 for RetGC. Our findings identify the functional interface on RD3 required for its inhibitory binding to RetGC, a process essential for protecting photoreceptors from degeneration.

GUCY2D mutations in retinal guanylyl cyclase 1 provide biochemical reasons for dominant cone-rod dystrophy but not for stationary night blindness.

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone-rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer-6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.

Regulation of retinal membrane guanylyl cyclase (RetGC) by negative calcium feedback and RD3 protein.

This article presents a brief overview of the main biochemical and cellular processes involved in regulation of cyclic GMP production in photoreceptors. The main focus is on how the fluctuations of free calcium concentrations in photoreceptors between light and dark regulate the activity of retinal membrane guanylyl cyclase (RetGC) via calcium sensor proteins. The emphasis of the review is on the structure of RetGC and guanylyl cyclase activating proteins (GCAPs) in relation to their functional role in photoreceptors and congenital diseases of photoreceptors. In addition to that, the structure and function of retinal degeneration-3 protein (RD3), which regulates RetGC in a calcium-independent manner, is discussed in detail in connections with its role in photoreceptor biology and inherited retinal blindness.

Retinal guanylyl cyclase activation by calcium sensor proteins mediates photoreceptor degeneration in an rd3 mouse model of congenital human blindness.

Deficiency of RD3 (retinal degeneration 3) protein causes recessive blindness and photoreceptor degeneration in humans and in the rd3 mouse strain, but the disease mechanism is unclear. Here, we present evidence that RD3 protects photoreceptors from degeneration by competing with guanylyl cyclase-activating proteins (GCAPs), which are calcium sensor proteins for retinal membrane guanylyl cyclase (RetGC). RetGC activity in rd3/rd3 retinas was drastically reduced but stimulated by the endogenous GCAPs at low Ca2+ concentrations. RetGC activity completely failed to accelerate in rd3/rd3GCAPs-/- hybrid photoreceptors, whose photoresponses remained drastically suppressed compared with the WT. However, ∼70% of the hybrid rd3/rd3GCAPs-/- photoreceptors survived past 6 months, in stark contrast to <5% in the nonhybrid rd3/rd3 retinas. GFP-tagged human RD3 inhibited GCAP-dependent activation of RetGC in vitro similarly to the untagged RD3. When transgenically expressed in rd3/rd3 mouse retinas under control of the rhodopsin promoter, the RD3GFP construct increased RetGC levels to near normal levels, restored dark-adapted photoresponses, and rescued rods from degeneration. The fluorescence of RD3GFP in rd3/rd3RD3GFP+ retinas was mostly restricted to the rod photoreceptor inner segments, whereas GCAP1 immunofluorescence was concentrated predominantly in the outer segment. However, RD3GFP became distributed to the outer segments when bred into a GCAPs-/- genetic background. These results support the hypothesis that an essential biological function of RD3 is competition with GCAPs that inhibits premature cyclase activation in the inner segment. Our findings also indicate that the fast rate of degeneration in RD3-deficient photoreceptors results from the lack of this inhibition.

Retinal degeneration protein 3 controls membrane guanylate cyclase activities in brain tissue.

The retinal degeneration protein RD3 is involved in regulatory processes of photoreceptor cells. Among its main functions is the inhibition of photoreceptor specific membrane guanylate cyclases during trafficking from the inner segment to their final destination in the outer segment. However, any physiological role of RD3 in non-retinal tissue is unsolved at present and specific protein targets outside of retinal tissue have not been identified so far. The family of membrane bound guanylate cyclases share a high homology of their amino acid sequences in their cytoplasmic domains. Therefore, we reasoned that membrane guanylate cyclases that are activated by natriuretic peptides are also regulated by RD3. We analyzed transcript levels of the rd3 gene and natriuretic peptide receptor genes Npr1 and Npr2 in the mouse retina, cerebellum, hippocampus, neocortex, and the olfactory bulb during development from the embryonic to the postnatal stage at P60. The rd3 gene showed a lower expression level than Npr1 and Npr2 (encoding for GC-A and GC-B, respectively) in all tested brain tissues, but was at least one order of magnitude higher in the retina. RD3 and natriuretic peptide receptor GCs co-express in the retina and brain tissue leading to functional tests. We expressed GC-A and GC-B in HEK293T cells and measured the inhibition of GCs by RD3 after activation by natriuretic peptides yielding inhibitory constants around 25 nM. Furthermore, endogenous GCs in astrocytes were inhibited by RD3 to a similar extent. We here show for the first time that RD3 can inhibit two hormone-stimulated GCs, namely GC-A and GC-B indicating a new regulatory feature of these hormone receptors.

Structural basis of retinal membrane guanylate cyclase regulation by GCAP1 and RD3.

Retinal membrane guanylate cyclases (RetGC1 and RetGC2) are expressed in photoreceptor rod and cone cells, where they promote the onset of visual recovery during phototransduction. The catalytic activity of RetGCs is regulated by their binding to regulatory proteins, guanylate cyclase activating proteins (GCAP1-5) and the retinal degeneration 3 protein (RD3). RetGC1 is activated by its binding to Ca2+-free/Mg2+-bound GCAP1 at low cytosolic Ca2+ levels in light-activated photoreceptors. By contrast, RetGC1 is inactivated by its binding to Ca2+-bound GCAP1 and/or RD3 at elevated Ca2+ levels in dark-adapted photoreceptors. The Ca2+ sensitive cyclase activation helps to replenish the cytosolic cGMP levels in photoreceptors during visual recovery. Mutations in RetGC1, GCAP1 or RD3 that disable the Ca2+-dependent regulation of cyclase activity are genetically linked to rod/cone dystrophies and other inherited forms of blindness. Here I review the structural interaction of RetGC1 with GCAP1 and RD3. I propose a two-state concerted model in which the dimeric RetGC1 allosterically switches between active and inactive conformational states with distinct quaternary structures that are oppositely stabilized by the binding of GCAP1 and RD3. The binding of Ca2+-free/Mg2+-bound GCAP1 is proposed to activate the cyclase by stabilizing RetGC1 in an active conformation (R-state), whereas Ca2+-bound GCAP1 and/or RD3 inhibit the cyclase by locking RetGC1 in an inactive conformation (T-state). Exposed hydrophobic residues in GCAP1 (residues H19, Y22, M26, F73, V77, W94) are essential for cyclase activation and could be targeted by rational drug design for the possible treatment of rod/cone dystrophies.

RNA-based therapies in animal models of Leber congenital amaurosis causing blindness.

Leber congenital amaurosis (LCA) is a severe, genetically heterogeneous recessive eye disease in which ~ 35% of gene mutations are in-frame nonsense mutations coding for loss-of-function premature termination codons (PTCs) in mRNA. Nonsense suppression therapy allows read-through of PTCs leading to production of full-length protein. A limitation of nonsense suppression is that nonsense-mediated decay (NMD) degrades PTC-containing RNA transcripts. The purpose of this study was to determine whether inhibition of NMD could improve nonsense suppression efficacy in vivo. Using a high-throughput approach in the recessive cep290 zebrafish model of LCA (cep290;Q1223X), we first tested the NMD inhibitor Amlexanox in combination with the nonsense suppression drug Ataluren. We observed reduced retinal cell death and improved visual function. With these positive data, we next investigated whether this strategy was also applicable across species in two mammalian models: Rd12 (rpe65;R44X) and Rd3 (rd3;R107X) mouse models of LCA. In the Rd12 model, cell death was reduced, RPE65 protein was produced, and in vivo visual function testing was improved. We establish for the first time that the mechanism of action of Amlexanox in Rd12 retina was through reduced UPF1 phosphorylation. In the Rd3 model, however, no beneficial effect was observed with Ataluren alone or in combination with Amlexanox. This variation in response establishes that some forms of nonsense mutation LCA can be targeted by RNA therapies, but that this needs to be verified for each genotype. The implementation of precision medicine by identifying better responders to specific drugs is essential for development of validated retinal therapies.

Optical coherence tomography and fundus autofluorescence imaging in an infant with RD3-related leber congenital amaurosis.

Background: Leber congenital amaurosis (LCA) is both genetically and phenotypically heterogeneous group of retinal disorder. Mutations in retinal degeneration 3 (RD3) have been reported as an infrequent cause of LCA which account for less than 1% of all known LCA cases. This case report provides Optical Coherence Tomography (OCT) and Fundus Autofluorescence (FAF) findings of an infant with LCA related to a mutation in RD3.Materials and Methods: Single retrospective case report.Results: TruSight One Expanded Sequencing Panel was applied to the patient on the Illumina NextSeq. Homozygous pathogenic variant (c.112 C > T, p.Arg38Ter) was detected in the RD3 gene. Well-demarcated central foveal atrophy was noted in the infrared imaging. FAF imaging showed perifoveal hyperautofluorescent ring and irregular hyperautofluorescence outside the vascular arcade. An arrest in foveal development and loss of outer retinal structure including outer nuclear layer, external limiting membrane, ellipsoid zone and interdigitation zone at the fovea were detected in the OCT imaging.Conclusion: This study indicates that RD3-related LCA has a very severe phenotype with foveal development arrest and very early loss of all photoreceptor layer and external limiting membrane at the fovea.

Control of the Nucleotide Cycle in Photoreceptor Cell Extracts by Retinal Degeneration Protein 3.

Retinal degeneration protein 3 (RD3) is crucial for photoreceptor cell survival and linked to Leber Congenital Amaurosis type 12 (LCA12), a hereditary retinal disease in humans. RD3 inhibits photoreceptor guanylate cyclases GC-E and GC-F and is involved in transport of GCs from the inner to the outer segments. Otherwise, its role in photoreceptor physiology is poorly understood. Here, we describe a new function of RD3. Purified RD3 evoked an increase in guanylate kinase activity, an enzyme that is involved in the nucleotide cycle in photoreceptors. We demonstrate a direct interaction between guanylate kinase and RD3 using back-scattering interferometry and show by immunohistochemistry of mouse retina sections that RD3 and guanylate kinase co-localize in photoreceptor inner segments and to a lesser extent in the outer plexiform layer. Our findings point toward a more complex function of RD3 in photoreceptors. The RD3 - guanylate kinase interaction may also play a role in other cellular systems, while the GC - RD3 interaction is exclusive to photoreceptors.

Chemical shift assignments of retinal degeneration 3 protein (RD3).

Retinal degeneration 3 protein (RD3) binds to retinal membrane guanylyl cyclase (RetGC) and suppresses the basal activity of RetGC in photoreceptor cells that opposes the allosteric activation of the cyclase by GCAP proteins. Mutations in RD3 that disrupt its inhibition of RetGC are implicated in human retinal degenerative disorders. Here we report both backbone and sidechain NMR assignments for the RD3 protein (BMRB accession no. 27305).

Photoreceptor Guanylate Cyclase (GUCY2D) Mutations Cause Retinal Dystrophies by Severe Malfunction of Ca2+-Dependent Cyclic GMP Synthesis.

Over 100 mutations in GUCY2D that encodes the photoreceptor guanylate cyclase GC-E are known to cause two major diseases: autosomal recessive Leber congenital amaurosis (arLCA) or autosomal dominant cone-rod dystrophy (adCRD) with a poorly understood mechanism at the molecular level in most cases. Only few mutations were further characterized for their enzymatic and molecular properties. GC-E activity is under control of neuronal Ca2+-sensor proteins, which is often a possible route to dysfunction. We investigated five recently-identified GC-E mutants that have been reported in patients suffering from arLCA (one large family) and adCRD/maculopathy (four families). Microsatellite analysis revealed that one of the mutations, c.2538G > C (p.K846N), occurred de novo. To better understand the mechanism by which mutations that are located in different GC-E domains develop different phenotypes, we investigated the molecular consequences of these mutations by expressing wildtype and mutant GC-E variants in HEK293 cells. Analyzing their general enzymatic behavior, their regulation by Ca2+ sensor proteins and retinal degeneration protein 3 (RD3) dimerization domain mutants (p.E841K and p.K846N) showed a shift in Ca2+-sensitive regulation by guanylate cyclase-activating proteins (GCAPs). Mutations in the cyclase catalytic domain led to a loss of enzyme function in the mutant p.P873R, but not in p.V902L. Instead, the p.V902L mutation increased the guanylate cyclase activity more than 20-fold showing a high GCAP independent activity and leading to a constitutively active mutant. This is the first mutation to be described affecting the GC-E catalytic core in a complete opposite way.

RD3 loss dictates high-risk aggressive neuroblastoma and poor clinical outcomes.

Clinical outcomes for high-risk neuroblastoma patients remains poor, with only 40-50% 5-Year overall survival (OS) and <10% long-term survival. The ongoing acquisition of genetic/molecular rearrangements in undifferentiated neural crest cells may endorse neuroblastoma progression. This study recognized the loss of Retinal Degeneration protein 3, RD3 in aggressive neuroblastoma, and identified its influence in better clinical outcomes and defined its novel metastasis suppressor function. The results showed ubiquitous expression of RD3 in healthy tissues, complete-loss and significant TNM-stage association of RD3 in clinical samples. RD3-loss was intrinsically associated with reduced OS, abridged relapse-free survival, aggressive stage etc., in neuroblastoma patient cohorts. RD3 was transcriptionally and translationally regulated in metastatic site-derived aggressive (MSDAC) cells (regardless of CSC status) ex vivo and in tumor manifolds from metastatic sites in reproducible aggressive disease models in vivo. Re-expressing RD3 in MSDACs reverted their metastatic potential both in vitro and in vivo. Conversely muting RD3 in neuroblastoma cells not only heightened invasion/migration but also dictated aggressive disease with metastasis. These results demonstrate the loss of RD3 in high-risk neuroblastoma, its novel, thus-far unrecognized metastasis suppressor function and further imply that RD3-loss may directly relate to tumor aggressiveness and poor clinical outcomes.