Three-dimensional architecture of the rod sensory cilium and its disruption in retinal neurodegeneration.

Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

Inherited Retinal Degeneration Caused by Dehydrodolichyl Diphosphate Synthase Mutation-Effect of an ALG6 Modifier Variant.

Modern advances in disease genetics have uncovered numerous modifier genes that play a role in the severity of disease expression. One such class of genetic conditions is known as inherited retinal degenerations (IRDs), a collection of retinal degenerative disorders caused by mutations in over 300 genes. A single missense mutation (K42E) in the gene encoding the enzyme dehydrodolichyl diphosphate synthase (DHDDS), which is required for protein N-glycosylation in all cells and tissues, causes DHDDS-IRD (retinitis pigmentosa type 59 (RP59; OMIM #613861)). Apart from a retinal phenotype, however, DHDDS-IRD is surprisingly non-syndromic (i.e., without any systemic manifestations). To explore disease pathology, we selected five glycosylation-related genes for analysis that are suggested to have disease modifier variants. These genes encode glycosyltransferases (ALG6, ALG8), an ER resident protein (DDOST), a high-mannose oligosaccharyl transferase (MPDU1), and a protein N-glycosylation regulatory protein (TNKS). DNA samples from 11 confirmed DHDDS (K42E)-IRD patients were sequenced at the site of each candidate genetic modifier. Quantitative measures of retinal structure and function were performed across five decades of life by evaluating foveal photoreceptor thickness, visual acuity, foveal sensitivity, macular and extramacular rod sensitivity, and kinetic visual field extent. The ALG6 variant, (F304S), was correlated with greater macular cone disease severity and less peripheral rod disease severity. Thus, modifier gene polymorphisms may account for a significant portion of phenotypic variation observed in human genetic disease. However, the consequences of the polymorphisms may be counterintuitively complex in terms of rod and cone populations affected in different regions of the retina.

Perspectives on Retinal Dolichol Metabolism, and Visual Deficits in Dolichol Metabolism-Associated Inherited Disorders.

De novo synthesis of dolichol (Dol) and dolichyl phosphate (Dol-P) is essential for protein glycosylation. Herein, we provide a brief overview of Dol and Dol-P synthesis and the maintenance of their cellular content. Retinal Dol metabolism and the requirement of Dol-linked oligosaccharide synthesis in the neural retina also are discussed. There are recently discovered and an emerging class of rare congenital disorders that affect Dol metabolism, involving the genes DHDDS, NUS1, SRD5A3, and DOLK. Further understanding of these congenital disorders is evolving, based upon studies utilizing yeast and murine models, as well as clinical reports of these rare disorders. We summarize the known visual deficits associated with Dol metabolism disorders, and identify the need for generation and characterization of suitable animal models of these disorders to elucidate the underlying molecular and cellular mechanisms of the associated retinopathies.

Vertebrate Animal Models of RP59: Current Status and Future Prospects.

Retinitis pigmentosa-59 (RP59) is a rare, recessive form of RP, caused by mutations in the gene encoding DHDDS (dehydrodolichyl diphosphate synthase). DHDDS forms a heterotetrameric complex with Nogo-B receptor (NgBR; gene NUS1) to form a cis-prenyltransferase (CPT) enzyme complex, which is required for the synthesis of dolichol, which in turn is required for protein N-glycosylation as well as other glycosylation reactions in eukaryotic cells. Herein, we review the published phenotypic characteristics of RP59 models extant, with an emphasis on their ocular phenotypes, based primarily upon knock-in of known RP59-associated DHDDS mutations as well as cell type- and tissue-specific knockout of DHDDS alleles in mice. We also briefly review findings in RP59 patients with retinal disease and other patients with DHDDS mutations causing epilepsy and other neurologic disease. We discuss these findings in the context of addressing "knowledge gaps" in our current understanding of the underlying pathobiology mechanism of RP59, as well as their potential utility for developing therapeutic interventions to block the onset or to dampen the severity or progression of RP59.

The B3 Subunit of the Cone Cyclic Nucleotide-gated Channel Regulates the Light Responses of Cones and Contributes to the Channel Structural Flexibility.

Cone photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in cone phototransduction, which is a process essential for daylight vision, color vision, and visual acuity. Mutations in the cone channel subunits CNGA3 and CNGB3 are associated with human cone diseases, including achromatopsia, cone dystrophies, and early onset macular degeneration. Mutations in CNGB3 alone account for 50% of reported cases of achromatopsia. This work investigated the role of CNGB3 in cone light response and cone channel structural stability. As cones comprise only 2-3% of the total photoreceptor population in the wild-type mouse retina, we used Cngb3(-/-)/Nrl(-/-) mice with CNGB3 deficiency on a cone-dominant background in our study. We found that, in the absence of CNGB3, CNGA3 was able to travel to the outer segments, co-localize with cone opsin, and form tetrameric complexes. Electroretinogram analyses revealed reduced cone light response amplitude/sensitivity and slower response recovery in Cngb3(-/-)/Nrl(-/-) mice compared with Nrl(-/-) mice. Absence of CNGB3 expression altered the adaptation capacity of cones and severely compromised function in bright light. Biochemical analysis demonstrated that CNGA3 channels lacking CNGB3 were more resilient to proteolysis than CNGA3/CNGB3 channels, suggesting a hindered structural flexibility. Thus, CNGB3 regulates cone light response kinetics and the channel structural flexibility. This work advances our understanding of the biochemical and functional role of CNGB3 in cone photoreceptors.

Overexpression of rod photoreceptor glutamic acid rich protein 2 (GARP2) increases gain and slows recovery in mouse retina.

BACKGROUND: The rod photoreceptor cGMP-gated cation channel, consisting of three α- and one ß subunit, controls ion flow into the rod outer segment (ROS). In addition to the ß-subunit, the Cngb1 locus encodes an abundant soluble protein, GARP2 that binds stoichiometrically to rod photoreceptor cGMP phosphodiesterase type 6 (PDE6). To examine the in vivo functional role of GARP2 we generated opsin promoter-driven transgenic mice overexpressing GARP2 three-fold specifically in rod photoreceptors. RESULTS: In the GARP2 overexpressing transgenic mice (tg), the endogenous channel ß-subunit, cGMP phosphodiesterase α-subunit, peripherin2/RDS and guanylate cyclase I were present at WT levels and were properly localized within the ROS. While localized properly within ROS, two proteins cGMP phosphodiesterase α-subunit (1.4-fold) and cGMP-gated cation channel α-subunit (1.2-fold) were moderately, but significantly elevated. Normal stratification of all retinal layers was observed, and ROS were stable in numbers but were 19% shorter than WT. Analysis of the photoresponse using electroretinography (ERG) showed that tg mice exhibit no change in sensitivity indicating overall normal rod function, however two parameters of the photoresponse significantly differed from WT responses. Fitting of the rising phase of the ERG a-wave to an accepted model of phototransduction showed a two-fold increase in phototransduction gain in the tg mice. The increase in gain was confirmed in isolated retinal tissue and by suction electrode recordings of individual rod photoreceptor cells. A measure of response recovery, the dominant time constant (τD) was elevated 69% in isolated retina compared to WT, indicating slower shutoff of the photoresponse. CONCLUSIONS: GARP2 may participate in regulating visual signal transduction through a previously unappreciated role in regulating phototransduction gain and recovery.

Cells Special Issue: "The Molecular and Cellular Basis of Retinal Diseases".

The recent success in the treatment of hereditary retinal disease caused by defects in the RPE65 gene and the FDA approval of this treatment has established the importance of the study of animal models and the translational impact of these research findings [...].

A Dhdds K42E knock-in RP59 mouse model shows inner retina pathology and defective synaptic transmission.

Retinitis pigmentosa (RP) defines a group of hereditary progressive rod-cone degenerations that exhibit a common phenotype caused by variants in over 70 genes. While most variants in the dehydrodolichyl diphosphate synthase (DHDDS) gene result in syndromic abnormalities, some variants cause non-syndromic RP (RP59). DHDDS encodes one subunit of the enzyme cis-prenyltransferase (CPT), which is required for the synthesis of dolichol (Dol), that is a necessary protein glycosylation cofactor. We previously reported the creation and initial characterization of a knock-in (KI) mouse model harboring the most prevalent RP59-associated DHDDS variant (K42E) to understand how defects in DHDDS lead to retina-specific pathology. This model exhibited no profound retinal degeneration, nor protein N-glycosylation defects. Here, we report that the Dol isoprenylogue species in retina, liver, and brain of the K42E mouse model are statistically shorter than in the corresponding tissues of age-matched controls, as reported in blood and urine of RP59 patients. Retinal transcriptome analysis demonstrated elevation of many genes encoding proteins involved in synaptogenesis and synaptic function. Quantitative retinal cell layer thickness measurements demonstrated a significant reduction in the inner nuclear layer (INL) and total retinal thickness (TRT) beginning at postnatal (PN) ∼2 months, progressively increasing to PN 18-mo. Histological analysis revealed cell loss in the INL, outer plexiform layer (OPL) disruption, and ectopic localization of outer nuclear layer (ONL) nuclei into the OPL of K42E mutant retinas, relative to controls. Electroretinograms (ERGs) of mutant mice exhibited reduced b-wave amplitudes beginning at PN 1-mo, progressively declining through PN 18-mo, without appreciable a-wave attenuation, relative to controls. Our results suggest that the underlying cause of DHDDS K42E variant driven RP59 retinal pathology is defective synaptic transmission from outer to inner retina.

Comparative intrinsic optical signal imaging of wild-type and mutant mouse retinas.

Functional measurement is important for retinal study and disease diagnosis. Transient intrinsic optical signal (IOS) response, tightly correlated with functional stimulation, has been previously detected in normal retinas. In this paper, comparative IOS imaging of wild-type (WT) and rod-degenerated mutant mouse retinas is reported. Both 2-month and 1-year-old mice were measured. In 2-month-old mutant mice, time course and peak value of the stimulus-evoked IOS were significantly delayed (relative to stimulus onset) and reduced, respectively, compared to age matched WT mice. In 1-year-old mutant mice, stimulus-evoked IOS was totally absent. However, enhanced spontaneous IOS responses, which might reflect inner neural remodeling in diseased retina, were observed in both 2-month and 1-year-old mutant retinas. Our experiments demonstrate the potential of using IOS imaging for noninvasive and high resolution identification of disease-associated retinal dysfunctions. Moreover, high spatiotemporal resolution IOS imaging may also lead to advanced understanding of disease-associated neural remodeling in the retina.

Age-related changes in Cngb1-X1 knockout mice: prolonged cone survival.

The rod photoreceptor cGMP-gated cation channel has an essential role in phototransduction functioning as the primary point for calcium and sodium entry into the rod outer segment. The channel consists of two subunits, α and ß. The α-subunit can function in isolation as an ion channel, and the ß-subunit modulates channel activity and has a structural role. We previously reported that a mouse knockout (KO) of the ß-subunit and related glutamic acid-rich proteins (GARPs) attenuates rod function and causes structural alterations and slowly progressive retinal degeneration. Here, we have extended our functional analyses of the KO mice evaluating rod and cone function using the electroretinogram in mice up to 4 months of age. Retinal stratification is preserved in the knockout mice at 3 months, and a significant number of cones remain up to 7 months based on PNA staining of cone sheaths. Electroretinography of KO mice at 1 month old revealed a diminished dark-adapted b-wave and normal light-adapted b-wave compared to wild-type mice. Over the next 3 months, both dark- and light-adapted b-wave amplitudes declined, but the reduction was greater for dark-adapted b-wave amplitudes. In one-month-old mice, the critical flicker frequency (CFF) was substantially lower for the KO mice at scotopic intensities, but normal at photopic intensities. CFF values remained stable in the KO mice as the b-wave amplitudes decreased with age. Declining b-wave amplitudes confirm an RP phenotype of rod followed by cone degeneration. Flicker responses show that the cone circuits function normally at threshold despite significant losses in the maximum light-adapted b-wave amplitude. These results confirm that rods are marginally functional in the absence of the ß-subunit and in addition show that CFF may be a more sensitive measure of remaining functional cone vision in animal models of RP undergoing progressive rod-cone degeneration.

Activation of retinal guanylyl cyclase RetGC1 by GCAP1: stoichiometry of binding and effect of new LCA-related mutations.

Retinal membrane guanylyl cyclase (RetGC) and Ca(2+)/Mg(2+) sensor proteins (GCAPs) control the recovery of the photoresponse in vertebrate photoreceptors, through their molecular interactions that remain rather poorly understood and controversial. Here we have determined the main RetGC isozyme (RetGC1):GCAP1 binding stoichiometry at saturation in cyto, using fluorescently labeled RetGC1 and GCAP1 coexpressed in HEK293 cells. In a striking manner, the equimolar binding of RetGC1 with GCAP1 in transfected HEK293 cells typical for wild-type RetGC1 was eliminated by a substitution, D639Y, in the kinase homology domain of RetGC1 found in a patient with a severe form of retinal dystrophy, Leber congenital amaurosis (LCA). A similar effect was observed with another LCA-related mutation, R768W, in the same domain of RetGC1. In contrast to the completely suppressed binding and activation of RetGC1 by Mg(2+)-liganded GCAP1, neither of these two mutations eliminated the GCAP1-independent activity of RetGC stimulated by Mn(2+). These results directly implicate the D639 (and possibly R768)-containing portion of the RetGC1 kinase homology domain in its primary recognition by the Mg(2+)-bound activator form of GCAP1.

Lack of Overt Retinal Degeneration in a K42E Dhdds Knock-In Mouse Model of RP59.

Dehydrodolichyl diphosphate synthase (DHDDS) is required for protein N-glycosylation in eukaryotic cells. A K42E point mutation in the DHDDS gene causes an autosomal recessive form of retinitis pigmentosa (RP59), which has been classified as a congenital disease of glycosylation (CDG). We generated K42E Dhdds knock-in mice as a potential model for RP59. Mice heterozygous for the Dhdds K42E mutation were generated using CRISPR/Cas9 technology and crossed to generate DhddsK42E/K42E homozygous mice. Spectral domain-optical coherence tomography (SD-OCT) was performed to assess retinal structure, relative to age-matched wild type (WT) controls. Immunohistochemistry against glial fibrillary acidic protein (GFAP) and opsin (1D4 epitope) was performed on retinal frozen sections to monitor gliosis and opsin localization, respectively, while lectin cytochemistry, plus and minus PNGase-F treatment, was performed to assess protein glycosylation status. Retinas of DhddsK42E/K42E mice exhibited grossly normal histological organization from 1 to 12 months of age. Anti-GFAP immunoreactivity was markedly increased in DhddsK42E/K42E mice, relative to controls. However, opsin immunolocalization, ConA labeling and PNGase-F sensitivity were comparable in mutant and control retinas. Hence, retinas of DhddsK42E/K42E mice exhibited no overt signs of degeneration, yet were markedly gliotic, but without evidence of compromised protein N-glycosylation. These results challenge the notion of RP59 as a DHDDS loss-of-function CDG and highlight the need to investigate unexplored RP59 disease mechanisms.

Selective Ablation of Dehydrodolichyl Diphosphate Synthase in Murine Retinal Pigment Epithelium (RPE) Causes RPE Atrophy and Retinal Degeneration.

Patients with certain defects in the dehydrodolichyl diphosphate synthase (DHDDS) gene (RP59; OMIM #613861) exhibit classic symptoms of retinitis pigmentosa, as well as macular changes, suggestive of retinal pigment epithelium (RPE) involvement. The DHDDS enzyme is ubiquitously required for several pathways of protein glycosylation. We wish to understand the basis for selective ocular pathology associated with certain DHDDS mutations and the contribution of specific ocular cell types to the pathology of mutant Dhdds-mediated retinal degeneration. To circumvent embryonic lethality associated with Dhdds knockout, we generated a Cre-dependent knockout allele of murine Dhdds (Dhddsflx/flx). We used targeted Cre expression to study the importance of the enzyme in the RPE. Structural alterations of the RPE and retina including reduction in outer retinal thickness, cell layer disruption, and increased RPE hyper-reflectivity were apparent at one postnatal month. At three months, RPE and photoreceptor disruption was observed non-uniformly across the retina as well as RPE transmigration into the photoreceptor layer, external limiting membrane descent towards the RPE, and patchy loss of photoreceptors. Functional loss measured by electroretinography was consistent with structural loss showing scotopic a- and b-wave reductions of 83% and 77%, respectively, at three months. These results indicate that RPE dysfunction contributes to DHDDS mutation-mediated pathology and suggests a more complicated disease mechanism than simply disruption of glycosylation.

Retinal Degeneration Caused by Rod-Specific Dhdds Ablation Occurs without Concomitant Inhibition of Protein N-Glycosylation.

Dehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step in dolichol synthesis. Recessive mutations in DHDDS cause retinitis pigmentosa (RP59), resulting in blindness. We hypothesized that rod photoreceptor-specific ablation of Dhdds would cause retinal degeneration due to diminished dolichol-dependent protein N-glycosylation. Dhddsflx/flx mice were crossed with rod-specific Cre recombinase-expressing (Rho-iCre75) mice to generate rod-specific Dhdds knockout mice (Dhddsflx/flx iCre+). In vivo morphological and electrophysiological evaluation of Dhddsflx/flx iCre+ retinas revealed mild retinal dysfunction at postnatal (PN) 4 weeks, compared with age-matched controls; however, rapid photoreceptor degeneration ensued, resulting in almost complete loss of rods and cones by PN 6 weeks. Retina dolichol levels were markedly decreased by PN 4 weeks in Dhddsflx/flx iCre+ mice, relative to controls; despite this, N-glycosylation of retinal proteins, including opsin (the dominant rod-specific glycoprotein), persisted in Dhddsflx/flx iCre+ mice. These findings challenge the conventional mechanistic view of RP59 as a congenital disorder of glycosylation.

GARP2 accelerates retinal degeneration in rod cGMP-gated cation channel ß-subunit knockout mice.

The Cngb1 locus-encoded ß-subunit of rod cGMP-gated cation channel and associated glutamic acid rich proteins (GARPs) are required for phototransduction, disk morphogenesis, and rod structural integrity. To probe individual protein structure/function of the GARPs, we have characterized several transgenic mouse lines selectively restoring GARPs on a Cngb1 knockout (X1-/-) mouse background. Optical coherence tomography (OCT), light and transmission electron microscopy (TEM), and electroretinography (ERG) were used to analyze 6 genotypes including WT at three and ten weeks postnatal. Comparison of aligned histology/OCT images demonstrated that GARP2 accelerates the rate of degeneration. ERG results are consistent with the structural analyses showing the greatest attenuation of function when GARP2 is present. Even 100-fold or more overexpression of GARP1 could not accelerate degeneration as rapidly as GARP2, and when co-expressed GARP1 attenuated the structural and functional deficits elicited by GARP2. These results indicate that the GARPs are not fully interchangeable and thus, likely have separate and distinct functions in the photoreceptor. We also present a uniform murine OCT layer naming nomenclature system that is consistent with human retina layer designations to standardize murine OCT, which will facilitate data evaluation across different laboratories.

Role of RDS and Rhodopsin in Cngb1-Related Retinal Degeneration.

PURPOSE: Rod photoreceptor outer segment (OS) morphogenesis, structural integrity, and proper signal transduction rely on critical proteins found in the different OS membrane domains (e.g., plasma, disc, and disc rim membrane). Among these key elements are retinal degeneration slow (RDS, also known as peripherin-2), rhodopsin, and the beta subunit of the cyclic nucleotide gated channel (CNGB1a), which have been found to interact in a complex. The purpose of this study was to evaluate the potential interplay between these three proteins by examining retinal disease phenotypes in animal models expressing varying amounts of CNGB1a, rhodopsin, and RDS. METHODS: Outer segment trafficking, retinal function, and photoreceptor structure were evaluated using knockout mouse lines. RESULTS: Eliminating Cngb1 and reducing RDS leads to additive defects in RDS expression levels and rod electroretinogram (ERG) function, (e.g., Cngb1-/-/rds+/- versus rds+/- or Cngb1-/-) but not to additive defects in rod ultrastructure. These additive effects also manifested in cone function: Photopic ERG responses were significantly lower in the Cngb1-/-/rds+/- versus rds+/- or Cngb1-/-, suggesting that eliminating Cngb1 can accelerate the cone degeneration that usually presents later in the rds+/-. This was not the case with rhodopsin; reducing rhodopsin levels in concert with eliminating CNGB1a did not lead to phenotypes more severe than those observed in the Cngb1 knockout alone. CONCLUSIONS: These data support a role for RDS as the core component of a multiprotein plasma membrane-rim-disc complex that has both a structural role in photoreceptor OS formation and maintenance and a functional role in orienting proteins for optimal signal transduction.

Varying the GARP2-to-RDS Ratio Leads to Defects in Rim Formation and Rod and Cone Function.

PURPOSE: The beta subunit of the rod cyclic nucleotide gated channel B1 (CNGB1) contains a proline/glutamic acid-rich N-terminal domain (GARP), which is also present in rods as a non-membrane-bound protein (GARP1/2). GARP2 and CNGB1 bind to retinal degeneration slow (RDS), which is present in the rims of rod and cone outer segment (OS) layers. Here we focus on the importance of RDS/GARP complexes in OS morphogenesis and stability. METHODS: Retinal structure, function, and biochemistry were assessed in GARP2-Tg transgenic mice crossed onto rds+/+, rds+/-, and rds-/- genetic backgrounds. RESULTS: GARP2 expression decreased in animals with reduced RDS levels. Overexpression of GARP2 led to abnormalities in disc stacking in GARP2-Tg/rds+/+ and the accumulation of abnormal vesicular structures in GARP2-Tg/rds+/- OS, as well as alterations in RDS-ROM-1 complex formation. These abnormalities were associated with diminished scotopic a- and b-wave amplitudes in GARP2-Tg mice on both the rds+/+ and rds+/- backgrounds. In addition, severe defects in cone function were observed in GARP2-Tg mice on all RDS backgrounds. CONCLUSIONS: Our results indicate that overexpression of GARP2 significantly exacerbates the defects in rod function associated with RDS haploinsufficiency and leads to further abnormalities in OS ultrastructure. These data also suggest that GARP2 expression in cones can be detrimental to cones. RDS/GARP interactions remain under investigation but are critical for both OS structure and function.

Characterization of 3',5' cyclic nucleotide phosphodiesterase activity in Y79 retinoblastoma cells: absence of functional PDE6.

PURPOSE: Previous studies identified rod photoreceptor cyclic GMP phosphodiesterase (PDE6) transcripts in the human Y79 retinoblastoma cell line. To assess the potential to utilize this cell line for structure/function studies of PDE6, we analyzed 3',5' cyclic nucleotide phosphodiesterase activity focusing on expression of PDE6. METHODS: DEAE-chromatography was used to fractionate PDE activity from Y79 cell homogenates. PCR was performed on cDNA generated from Y79 cells and retina with PDE isoform specific primers. Western blots were performed with antibodies to PDE1, PDE4, or rod PDE6. DNA sequencing and protein truncation tests were performed with plasmids containing the entire coding region of Y79 rod PDE6 transcripts. Proteasome mediated degradation of PDE6 subunits was analyzed with a pathway specific inhibitor. Polysome isolation was performed by fractionation on sucrose gradients followed by RT-PCR for the PDE6 transcripts. RESULTS: Of three peaks of PDE activity, peaks 1 and 2 were activated by Ca2+/calmodulin, inhibited by dipyridamole and zaprinast, and were reactive with a PDE1 antibody. Peak 3 hydrolyzed only cAMP and was rolipram sensitive, indicative of PDE4. Transcripts for rod and cone PDE6 isoforms were detected in Y79 total RNA, however PDE6 antibodies recognized only a single 99 kDa polypeptide from immunoprecipitated 35S labeled Y79 extracts. DNA sequencing of PDE6 alpha, beta, gamma, and PDE6 associated delta-subunit cDNA revealed some polymorphism, but no apparent mutations. Each of the PDE6 transcripts could be translated into protein of the correct length. The concentration of cGMP in the cells was greatly reduced in comparison to that reported in the photoreceptor cell. Addition of cyclic nucleotide analogues, zinc, or butyrate did not enhance the expression of PDE6. Transduction into Y79 cells of adenovirus expressing PDE6 subunits failed to produce functional enzyme CONCLUSIONS: PDE1 and PDE4 enzyme activities predominate in Y79 cells. Despite the presence of PDE6 transcripts and the ability to translate each into protein in vitro, a functional PDE6 enzyme could not be detected. Attempts to enhance expression with cell culture or with introduction of virus expressing PDE6 were not successful. The results indicate that expression of a fully active stable PDE6 enzyme requires other post-transcriptional events that do not occur or are inhibited in Y79 cells.

Electroretinographic abnormalities in parents of patients with Leber congenital amaurosis who have heterozygous GUCY2D mutations.

BACKGROUND: Leber congenital amaurosis (LCA) is an infrequently encountered congenital form of retinitis pigmentosa with marked genetic and clinical heterogeneity. Thus far, 10 genes have been identified in this disorder since 1996. In the future, LCA may become treatable by gene and/or pharmacological intervention, and these therapies will likely be gene specific, giving major significance to rapid gene identification and gene-phenotype studies. OBJECTIVE: To test the hypothesis that parents of patients with LCA have identifiable electroretinographic and psychophysical changes. SUBJECTS, MATERIALS, AND METHODS: Complete eye examinations and electroretinographic studies were performed on 2 sets of parents whose offspring were diagnosed as having LCA and who were found to carry a mutation in 1 of the 10 LCA genes-GUCY2D. One set of parents also underwent static perimetry threshold measurements. RESULTS: We found that single flash-light-adapted a- and b-wave amplitudes, 30-Hz flicker, or both cone signals were significantly decreased in amplitude in 4 heterozygotes, while 2 parents showed delayed 30-Hz flicker implicit times. Electroretinographic rod-mediated signals were normal in 2 of the heterozygotes, but subnormal in 2. Static perimetry testing showed normal thresholds in the 2 heterozygotes tested. MAIN OUTCOME MEASURES: Single flash-light-adapted a- and b-wave amplitudes and implicit times, 30- or 32-Hz flicker amplitudes and implicit times, rod-mediated signals, and dark-adapted, rod-mediated thresholds. CONCLUSIONS: Some carrier parents of patients with LCA and a GUCY2D mutation develop measurable, cone and possibly rod abnormalities most consistent with a mild cone-rod dysfunction. This correlates well with the known retinal expression pattern of GUCY2D, which is considerably higher in cone compared with rod photoreceptor cells.

Dynamic near-infrared imaging reveals transient phototropic change in retinal rod photoreceptors.

Stiles-Crawford effect (SCE) is exclusively observed in cone photoreceptors, but why the SCE is absent in rod photoreceptors is still a mystery. In this study, we employed dynamic near infrared light imaging to monitor photoreceptor kinetics in freshly isolated frog and mouse retinas stimulated by oblique visible light flashes. It was observed that retinal rods could rapidly (onset: ∼10 ms for frog and 5 ms for mouse; time-to-peak: ∼200 ms for frog and 30 ms for mouse) shift toward the direction of the visible light, which might quickly compensate for the loss of luminous efficiency due to oblique illumination. In contrast, such directional movement was negligible in retinal cones. Moreover, transient rod phototropism could contribute to characteristic intrinsic optical signal (IOS). We anticipate that further study of the transient rod phototropism may not only provide insight into better understanding of the nature of vision but also promise an IOS biomarker for functional mapping of rod physiology at high resolution.