Characterization of RPE65 and RDH12, Two Enzymes Associated with Retinal Dystrophy and Retinoid Processing.

Phototransduction in vertebrate vision is mediated by visual pigments composed of opsin apoproteins covalently attached to a light-sensitive chromophore, 11-cis retinal. Absorption of light isomerizes 11-cis retinal to all-trans retinal, initiating a signal transduction cascade. A complex set of enzyme reactions occurring in the retinal pigment epithelium and the retina is responsible for the synthesis and regeneration of the chromophore and is termed the visual cycle. Several forms of inherited retinal degeneration and dysfunction manifest as a result of mutations in the genes associated with visual cycle function, such as RPE65 and RDH12. RPE65 is essential for the synthesis of 11-cis retinal and was recently confirmed to function as the visual cycle isomerase. Cone photoreceptors have been proposed to possess an exclusive chromophore regenerative pathway. Using a monoclonal antibody approach, we have now mapped antigenic determinants of the protein surface, shown that RPE65 is not expressed in cone cells, and confirmed that RPE65 is associated with the visual cycle enzyme RDH5. Rdh12 has an in vitro activity and localization profile that made it an excellent candidate to serve as the all-trans retinal reductase of the visual cycle. Immunochemical analysis localized RDH12 protein to the photoreceptor inner segments and outer nuclear layer in both humans and mice, suggesting an equivalent physiological role for RDH12 in both species. However, analysis of the phenotype of Rdh12-deficient mice revealed no differences in histology, retinoid processing or electroretinogram response compared to wild-type. Rdh12-deficient mice did show a decreased ability to reduce all-trans retinal and 11-cis retinal as measured by in vitro activity assays of retinal homogenates. These findings suggest that RDH12 function in mice does not directly contribute to visual cycle function. Instead, a critical function of RDH12 is likely the reduction of retinaldehydes that exceed the reductive capacity of the photoreceptor outer segment and gain access to the inner segments in conditions of high illumination. The study of these genes is important not only to gain a better understanding of visual cycle mechanism, but also to elucidate mechanisms of pathogenesis and to develop targeted forms of therapeutic intervention.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/76008/1/jareddc_1.pd

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals

Rdh12 Activity and Effects on Retinoid Processing in the Murine Retina*

RDH12 mutations are responsible for early-onset autosomal recessive retinal dystrophy, which results in profound retinal pathology and severe visual handicap in patients. To investigate the function of RDH12 within the network of retinoid dehydrogenases/reductases (RDHs) present in retina, we studied the retinal phenotype of Rdh12-deficient mice. In vivo rates of all-trans-retinal reduction and 11-cis-retinal formation during recovery from bleaching were similar in Rdh12-deficient and wild-type mice matched for an Rpe65 polymorphism that impacts visual cycle efficiency. However, retinal homogenates from Rdh12-deficient mice exhibited markedly decreased capacity to reduce exogenous retinaldehydes in vitro. Furthermore, in vivo levels of the bisretinoid compound diretinoid-pyridinium-ethanolamine (A2E) were increased in Rdh12-deficient mice of various genetic backgrounds. Conversely, in vivo levels of retinoic acid and total retinol were significantly decreased. Rdh12 transcript levels in wild-type mice homozygous for the Rpe65-Leu450 polymorphism were greater than in Rpe65-Met450 mice and increased during postnatal development in wild-type mice and Nrl-deficient mice having an all-cone retina. Rdh12-deficient mice did not exhibit increased retinal degeneration relative to wild-type mice at advanced ages, when bred on the light-sensitive BALB/c background, or when heterozygous for a null allele of superoxide dismutase 2 (Sod2+/−). Our findings suggest that a critical function of RDH12 is the reduction of all-trans-retinal that exceeds the reductive capacity of the photoreceptor outer segments