David A. Saperstein

Atomic-force microscopy: Rhodopsin dimers in native disc membranes

In vertebrate retinal photoreceptors, the rod outer-segment disc membranes contain densely packed rhodopsin molecules for optimal light absorption and subsequent amplification by the visual signalling cascade, but how these photon receptors are organized with respect to each other is not known. Here we use infrared-laser atomic-force microscopy to reveal the native arrangement of rhodopsin, which forms paracrystalline arrays of dimers in mouse disc membranes. The visualization of these closely packed rhodopsin dimers in native membranes gives experimental support to earlier inferences about their supramolecular structure and provides insight into how light signalling is controlled.

Organization of the G Protein-coupled Receptors Rhodopsin and Opsin in Native Membranes

G protein-coupled receptors (GPCRs), which constitute the largest and structurally best conserved family of signaling molecules, are involved in virtually all physiological processes. Crystal structures are available only for the detergent-solubilized light receptor rhodopsin. In addition, this receptor is the only GPCR for which the presumed higher order oligomeric state in native membranes has been demonstrated (Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., and Palczewski, K. (2003) Nature 421, 127–128). Here, we have determined by atomic force microscopy the organization of rhodopsin in native membranes obtained from wild-type mouse photoreceptors and opsin isolated from photoreceptors of Rpe65–/– mutant mice, which do not produce the chromophore 11-cis-retinal. The higher order organization of rhodopsin was present irrespective of the support on which the membranes were adsorbed for imaging. Rhodopsin and opsin form structural dimers that are organized in paracrystalline arrays. The intradimeric contact is likely to involve helices IV and V, whereas contacts mainly between helices I and II and the cytoplasmic loop connecting helices V and VI facilitate the formation of rhodopsin dimer rows. Contacts between rows are on the extracellular side and involve helix I. This is the first semi-empirical model of a higher order structure of a GPCR in native membranes, and it has profound implications for the understanding of how this receptor interacts with partner proteins.

Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies

Inherited retinopathies are a genetically and phenotypically heterogeneous group of diseases affecting approximately one in 2000 individuals worldwide. For the past 10 years, the Laboratory for Molecular Diagnosis of Inherited Eye Diseases (LMDIED) at the University of Texas‐Houston Health Science Center has screened subjects ascertained in the United States and Canada for mutations in genes causing dominant and recessive autosomal retinopathies. A combination of single strand conformational analysis (SSCA) and direct sequencing of five genes (rhodopsin, peripherin/RDS, RP1, CRX, and AIPL1) identified the disease‐causing mutation in approximately one‐third of subjects with autosomal dominant retinitis pigmentosa (adRP) or with autosomal dominant cone‐rod dystrophy (adCORD). In addition, the causative mutation was identified in 15% of subjects with Leber congenital amaurosis (LCA). Overall, we report identification of the causative mutation in 105 of 506 (21%) of unrelated subjects (probands) tested; we report five previously unreported mutations in rhodopsin, two in peripherin/RDS, and one previously unreported mutation in the cone‐rod homeobox gene, CRX. Based on this large survey, the prevalence of disease‐causing mutations in each of these genes within specific disease categories is estimated. These data are useful in estimating the frequency of specific mutations and in selecting individuals and families for mutation‐specific studies. Hum Mutat 17:42–51, 2001.

The G protein-coupled receptor rhodopsin in the native membrane

The higher‐order structure of G protein‐coupled receptors (GPCRs) in membranes may involve dimerization and formation of even larger oligomeric complexes. Here, we have investigated the organization of the prototypical GPCR rhodopsin in its native membrane by electron and atomic force microscopy (AFM). Disc membranes from mice were isolated and observed by AFM at room temperature. In all experimental conditions, rhodopsin forms structural dimers organized in paracrystalline arrays. A semi‐empirical molecular model for the rhodopsin paracrystal is presented validating our previously reported results. Finally, we compare our model with other currently available models describing the supramolecular structure of GPCRs in the membrane.

Role of Photoreceptor-specific Retinol Dehydrogenase in the Retinoid Cycle in Vivo

The retinoid cycle is a recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH–/–) mouse. Histological analysis and electron microscopy of retinas from 6- to 8-week-old prRDH–/– mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-cis-retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.

Characterization of retinal guanylate cyclase-activating protein 3 (GCAP3) from zebrafish to man

Calmodulin‐like neuronal Ca2+‐binding proteins (NCBPs) are expressed primarily in neurons and contain a combination of four functional and nonfunctional EF‐hand Ca2+‐binding motifs. The guanylate cyclase‐activating proteins 1–3 (GCAP1–3), the best characterized subgroup of NCBPs, function in the regulation of transmembrane guanylate cyclases 1–2 (GC1–2). The pairing of GCAPs and GCs in vivo depends on cell expression. Therefore, we investigated the expression of these genes in retina using in situ hybridization and immunocytochemistry. Our results demonstrate that GCAP1, GCAP2, GC1 and GC2 are expressed in human rod and cone photoreceptors, while GCAP3 is expressed exclusively in cones. As a consequence of extensive modification, the GCAP3 gene is not expressed in mouse retina. However, this lack of evolutionary conservation appears to be restricted to only some species as we cloned all three GCAPs from teleost (zebrafish) retina and localized them to rod cells, short single cones (GCAP1–2), and all subtypes of cones (GCAP3). Furthermore, sequence comparisons and evolutionary trace analysis coupled with functional testing of the different GCAPs allowed us to identify the key conserved residues that are critical for GCAP structure and function, and to define class‐specific residues for the NCBP subfamilies.

Oral 9-cis retinoid for childhood blindness due to Leber congenital amaurosis caused by RPE65 or LRAT mutations: an open-label phase 1b trial

BACKGROUND Leber congenital amaurosis, caused by mutations in RPE65 and LRAT, is a severe form of inherited retinal degeneration leading to blindness. We aimed to assess replacement of the missing chromophore 11-cis retinal with oral QLT091001 (synthetic 9-cis-retinyl acetate) in these patients. METHODS In our open-label, prospective, phase 1b trial, we enrolled patients (aged ≥6 years) with Leber congenital amaurosis and RPE65 or LRAT mutations at McGill Universitys Montreal Childrens Hospital. Patients received 7 days of oral QLT091001 (10-40 mg/m(2) per day). We assessed patients at baseline and days 7, 9, 14, and 30, and then 2 months and every 2 months thereafter for up to 2·2 years for safety outcomes and visual function endpoints including Goldmann visual fields (GVF), visual acuity, and functional MRI assessment. We regarded patients as having an improvement in vision if we noted at least a 20% improvement in retinal area on GVF compared with baseline or a visual acuity improvement of five or more letters compared with baseline in two consecutive study visits (or any improvement from no vision at baseline). This study is registered with ClinicalTrials.gov, number NCT01014052. FINDINGS Between December, 2009, and June, 2011, we enrolled and treated 14 patients aged 6-38 years who were followed up until March, 2012. Ten (71%) of 14 patients had an improvement in GVF areas (mean increase in retinal area of 28-683%). Six (43%) patients had an improvement in visual acuity (mean increase of 2-30 letters). Self-reported or parent-reported improvements in activities of daily living supported these findings. After 2 years, 11 (79%) patients had returned to their baseline GVF retinal area and ten (71%) had returned to baseline visual acuity letter values. Thus, three (21%) patients had a sustained GVF response and four (30%) had a sustained visual acuity response. Four patients had functional MRI scans, which correlated with visual response or absence of response to treatment. No serious adverse events occurred, although we noted transient headaches (11 patients), photophobia (11 patients), reduction in serum HDL concentrations (four patients), and increases in serum triglycerides (eight patients) and aspartate aminotransferase concentrations (two patients). INTERPRETATION Non-invasive oral QLT091001 therapy is well tolerated, and can rapidly improve visual function in some patients with Leber congenital amaurosis and RPE65 and LRAT mutations. FUNDING QLT, Foundation Fighting Blindness Canada, CIHR, FRSQ, Reseau Vision.

Novel RDH12 mutations associated with Leber congenital amaurosis and cone-rod dystrophy: biochemical and clinical evaluations.

The purpose of this study was to determine the role of the retinol dehydrogenase 12 (RDH12) gene in patients affected with Leber congenital amaurosis (LCA), autosomal recessive retinitis pigmentosa (arRP) and autosomal dominant/recessive cone-rod dystrophies (CORD). Changes in the promoter region, coding regions and exon/intron junctions of the RDH12 gene were evaluated using direct DNA sequencing of patients affected with LCA (n=36 cases), RP (n=62) and CORD (n=21). The allele frequency of changes observed was assessed in a multiethnic control population (n=159 individuals). Detailed biochemical and structural modeling analysis of the observed mutations were performed to assess their biological role in the inactivation of Rdh12. A comprehensive clinical assessment of retinal structure and function in LCA patients carrying mutations in the RDH12 gene was completed. Of the six changes identified, three were novel including a homozygous C201R change in a patient affected with LCA, a heterozygous A177V change in patients affected with CORD and a heterozygous G46G change in a patient affected with LCA. A novel compound heterozygote T49M/A269fsX270 mutation was also found in a patient with LCA, and both homozygous and heterozygous R161Q changes were seen in 26 patients affected with LCA, CORD or RP. These R161Q, G46G and the A177V sequence changes were shown to be polymorphic. We found that Rdh12 mutant proteins associated with LCA were inactive or displayed only residual activity when expressed in COS-7 and Sf9 cells, whereas those mutants that were considered polymorphisms were fully active. Thus, impairment of retinal structure and function for patients carrying these mutations correlated with the biochemical properties of the mutants.

Rhodopsin dimers in native disc membranes

In vertebrate retinal photoreceptors, the rod outer-segment disc membranes contain densely packed rhodopsin molecules for optimal light absorption and subsequent amplification by the visual signalling cascade, but how these photon receptors are organized with respect to each other is not known. Here we use infrared-laser atomic-force microscopy to reveal the native arrangement of rhodopsin, which forms paracrystalline arrays of dimers in mouse disc membranes. The visualization of these closely packed rhodopsin dimers in native membranes gives experimental support to earlier inferences about their supramolecular structure and provides insight into how light signalling is controlled.

Effects of Long-Term Administration of 9-cis-Retinyl Acetate on Visual Function in Mice

PURPOSE Long-term effects of treatment with 9-cis-retinyl acetate (9-cis-R-Ac), an artificial retinoid prodrug, were tested on changes in rod and cone visual functions in mice. METHODS The acetyl ester of the functional geometric chromophore 9-cis-retinal was delivered by oral gavage to C57BL/6 female mice. In initial experiments, 10-month-old mice were used for the single treatment with 9-cis-R-Ac or the control vehicle. In long-term experiments, 4-month-old mice were treated with 9-cis-R-Ac monthly for 6 and 10 months. Photoreceptor status was evaluated by various electroretinographic (ERG) techniques, retinoid analyses, and retinal morphology. Opsin, the predicted target of oxidized 9-cis-R-Ac, was purified and its chromophore was characterized. RESULTS Age-related changes observed in vehicle-treated mice at 10 months of age, compared with those in 4-month-old mice, included a progressive decline in ERG responses, such as a decreased rate of dark adaptation and a lowered rhodopsin/opsin ratio. Administration of 9-cis-R-Ac increased the rhodopsin regeneration ratio, and improved ERG responses and dark adaptation. Compared with vehicle-treated control animals, 10- and 14-month-old mice treated monthly with 9-cis-R-Ac for 6 or 10 months exhibited improved dark adaptation. In 14-month-old mice treated monthly, changes in the expression of retina-specific genes in the eye were detected by mRNA expression profiling, but no significant effects in gene expression were detected in the liver and kidney. CONCLUSIONS Deteriorating photoreceptor function documented in mice at 10 and 14 versus 4 months of age was improved significantly by long-term, monthly administration of 9-cis-R-Ac. These findings suggest a potential therapeutic approach to prevent age-related retinal dysfunction.