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T M Redmond

Publications and source records attributed to T M Redmond.

At least 19 recordsLinked to original sources
PubMed2003

Entrainment of circadian rhythm to a photoperiod reversal shows retinal dystrophy in RPE65(-/-) mice.

Light entrainment of circadian rhythms is mediated by classical "visual" photoreceptors (rods and cones) as well as "nonvisual" photoreceptive elements (light-detecting cells that do not contribute to classical "vision"). This paper aimed to assess whether light entrainment of locomotor circadian rhythms in mice with impaired rods and cones differs from normal controls and whether this technique, alongside existing techniques, could be used to assess visual function. The study was primarily interested in differences between the entrainment of circadian rhythms of normal-sighted C57Bl/6J mouse and the C57Bl/RPE65 knockout mouse (RPE65(-/-)), although C3H/HeJ (rd/rd) mice were included as a preexisting model of retinal degeneration. Circadian rhythms of motor activity before and after a 12-h light reversal were assessed in custom-built cages that continuously monitored movement. The controls showed a significantly higher mesor and amplitude when compared to the RPE65(-/-) and rd/rd mice. Despite the loss of rods and cones, the RPE65(-/-) and rd/rd maintained a 24-h circadian rhythm entrained to light similar to controls and were capable of circadian reentrainment to a 12-h light reversal. Importantly, this light reentrainment of the circadian phase occurred at a significantly slower rate in the retinal degenerate models than in the controls. The RPE65(-/-) model demonstrates a retinal degenerate reentrainment phenotype when compared to the rd/rd model. It is suggested that these retinal degenerate mice retain the ability to detect light for the purposes of circadian rhythm entrainment. However, alterations of specific parameters of the circadian rhythm with loss of rods and cones may provide measures of loss of visual function (sight).

Activity Cycles↗
PubMed2001

Sequence and structure of the mouse gene for RPE65.

PURPOSE: To determine the genomic organization of the mouse gene for the retinal pigment epithelium (RPE) specific protein RPE65. METHODS: A genomic clone containing the entire Rpe65 gene was isolated from a mouse genomic P1 library. Fragments of this clone were subcloned and sequenced by automated fluorescent dideoxy DNA sequencing and analyzed. Direct sequencing of PCR amplification products was used to complete the structure. Primer extension analysis was used to determine the transcription start site. RESULTS: Southern hybridization of restriction digests of mouse genomic DNA reveals a likely single autosomal gene for Rpe65 with no evidence of pseudogenes. Sequence analysis of the mouse P1 clone for Rpe65 and fragments thereof reveals 14 exons distributed over 27 kbp. The transcription start site is located 57 bp upstream of the initiation codon. The protein encoded by the mouse Rpe65 gene is highly conserved when compared with RPE65s from other species. CONCLUSIONS: RPE65 is a highly conserved protein and it appears that the genes for the mouse and human RPE65s, at least, are also conserved in overall structure.

Amino Acid Sequence↗
PubMed2001

Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinal pigment epithelium.

Choroidal neovascularization in age-related macular degeneration is a frequent and poorly treatable cause of vision loss in elderly Caucasians. This choroidal neovascularization has been associated with the expression of vascular endothelial growth factor (VEGF). In current animal models choroidal neovascularization is induced by subretinal injection of growth factors or vectors encoding growth factors such as VEGF, or by disruption of the Bruch's membrane/retinal pigment epithelium complex with laser treatment. We wished to establish a transgenic murine model of age-related macular degeneration, in which the overexpression of VEGF by the retinal pigment epithelium induces choroidal neovascularization. A construct consisting of a tissue-specific murine retinal pigment epithelium promoter (RPE(65) promoter) coupled to murine VEGF(164) cDNA with a rabbit beta-globin-3' UTR was introduced into the genome of albino mice. Transgene mRNA was expressed in the retinal pigment epithelium at all ages peaking at 4 months. The expression of VEGF protein was increased in both the retinal pigment epithelium and choroid. An increase of intravascular adherent leukocytes and vessel leakage was observed. Histopathology revealed intrachoroidal neovascularization that did not penetrate through an intact Bruch's membrane. These results support the hypothesis that additional insults to the integrity of Bruch's membrane are required to induce growth of choroidal vessels into the subretinal space as seen in age-related macular degeneration. This model may be useful to screen for inhibitors of choroidal vessel growth.

Age Factors↗
PubMed2001

New views on RPE65 deficiency: the rod system is the source of vision in a mouse model of Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is the most serious form of the autosomal recessive childhood-onset retinal dystrophies. Mutations in the gene encoding RPE65, a protein vital for regeneration of the visual pigment rhodopsin in the retinal pigment epithelium, account for 10-15% of LCA cases. Whereas previous studies of RPE65 deficiency in both animal models and patients attributed remaining visual function to cones, we show here that light-evoked retinal responses in fact originate from rods. For this purpose, we selectively impaired either rod or cone function in Rpe65-/- mice by generating double- mutant mice with models of pure cone function (rhodopsin-deficient mice; Rho-/-) and pure rod function (cyclic nucleotide-gated channel alpha3-deficient mice; Cnga3-/-). The electroretinograms (ERGs) of Rpe65-/- and Rpe65-/-Cnga3-/- mice were almost identical, whereas there was no assessable response in Rpe65-/-Rho-/- mice. Thus, we conclude that the rod system is the source of vision in RPE65 deficiency. Furthermore, we found that lack of RPE65 enables rods to mimic cone function by responding under normally cone-isolating lighting conditions. We propose as a mechanism decreased rod sensitivity due to a reduction in rhodopsin content to less than 1%. In general, the dissection of pathophysiological processes in animal models through the introduction of additional, selective mutations is a promising concept in functional genetics.

Animals↗
PubMed2001

Expression, purification, and MALDI analysis of RPE65.

PURPOSE: RPE65 is preferentially expressed in the retinal pigment epithelium (RPE) and is essential for retinal function. The purpose of the study was to develop methods for the expression of the protein, determine the accurate molecular weight of this expressed protein, and quantitate the amount of RPE65 in the bovine RPE. METHODS: Human RPE65 was expressed in Sf9 cells using the baculovirus system. The subcellular localization was determined by Western blot analysis and immunocytochemistry. An ELISA was developed for RPE65 and used to measure levels in bovine RPE. Recombinant and native RPE65 were purified by affinity chromatography. Molecular mass was determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. RESULTS: Recombinant human (rH)RPE65 was expressed as a major protein associated with cell membrane in Sf9 cells. The recombinant protein was purified to apparent homogeneity from both the membrane and nonmembrane fractions. The identity of the purified protein was confirmed by Western blot analysis and by partial peptide sequencing. rHRPE65 from the nonmembrane fraction has a mass of 64,867 +/- 80 which is close to the calculated molecular weight from the amino acid sequence including the His-tag (64,663), whereas the membrane-associated rHRPE65 has a molecular mass of 65,380 +/- 150, which is significantly higher than that of the non-membrane-associated form and the calculated molecular weight, suggesting posttranslational modifications. Similarly, native RPE65 was detected in the cytosolic and microsomal fractions of the bovine RPE, with an average level of 3.8 +/- 1.3 and 7.2 +/- 0.4 microg RPE65 per eye, respectively. The cytosolic form had a molecular mass of 61,161 +/- 60, which is close to the calculated value (60,944), whereas that of the microsomal form was 61,961 +/- 170. CONCLUSIONS: RPE65 is expressed in two forms, one of which is membrane associated and contains significant posttranslational modifications, similar to the native membrane-associated form.

Animals↗
PubMed2001

Effect of Rpe65 knockout on accumulation of lipofuscin fluorophores in the retinal pigment epithelium.

PURPOSE: In all mammalian species examined to date the retinal pigment epithelium (RPE) has been found to accumulate autofluorescent lysosomal storage bodies (lipofuscin) during senescence. Substantial evidence indicates that retinoids in the RPE-retina complex play a major role in RPE lipofuscin formation. Indeed, at least one RPE lipofuscin fluorophore is derived in part from vitamin A aldehyde. However, the precise mechanisms by which retinoids modulate RPE lipofuscin accumulation have not been elucidated. In mice without a functional Rpe65 gene, isomerization of all-trans- to 11-cis-retinol is blocked. Experiments were performed to determine whether this impairment of retinoid metabolism alters RPE lipofuscin accumulation. METHODS: RPE lipofuscin fluorophore content was compared in 12- to 13-month-old Rpe65(+/+), Rpe65(+/-), and Rpe65(-/-) mice. Lipofuscin fluorophore content was determined using quantitative fluorometric measurements. RPE lipofuscin content was also estimated with quantitative ultrastructural techniques. RESULTS: In the Rpe65(-/-) mice, RPE lipofuscin fluorophore accumulation was almost abolished. In addition, a significantly reduced accumulation of lipofuscin fluorophores was also observed in the Rpe65(+/-) animals. The inability of the RPE of)Rpe65(-/-) mice to supply 11-cis-retinal from the RPE to the retinal photoreceptors was accompanied by a massive accumulation of lipid droplets in the RPE that appeared to contain substantial amounts of retinoids. CONCLUSIONS: These findings indicate that formation of RPE lipofuscin fluorophores is almost completely dependent on a normal visual cycle. The absence of retinal (both all-trans and 11-cis) in Rpe65 knockout mice drastically reduced formation of lipofuscin fluorophores in these animals. Even an excessive accumulation of retinyl fatty acid esters in the RPE of Rpe65 knockout mice did not contribute to lipofuscin accumulation.

Animals↗
PubMed2000

Identification, expression, and substrate specificity of a mammalian beta-carotene 15,15'-dioxygenase.

We have identified from mouse the first mammalian beta-carotene 15,15'-dioxygenase (beta-CD), a crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived precursors. beta-CD is related to the retinal pigment epithelium-expressed protein RPE65 and belongs to a diverse family that includes the plant 9-cis-epoxycarotenoid dioxygenase and bacterial lignostilbene dioxygenases. beta-CD expression in Escherichia coli cells engineered to produce beta-carotene led to the accumulation of all-trans-retinal at the expense of beta-carotene, confirming that beta-CD catalyzed the central cleavage of this vitamin A precursor. Purified recombinant beta-CD protein cleaves beta-carotene in vitro with a V(max) of 36 pmol of retinal/mg of enzyme/min and a K(m) of 6 microm. Non-provitamin A carotenoids were also cleaved, although with much lower activity. By Northern analysis, a 2.4-kilobase (kb) message was observed in liver, kidney, small intestine, and testis, tissues important in retinoid/carotenoid metabolism. This message encoded a 63-kDa cytosolic protein expressed in these tissues. A shorter transcript of 1.8 kb was found in testis and skin. Developmentally, the 2.4-kb mRNA was abundant at embryonic day 7, with lower expression at embryonic days 11, 13, and 15, suggesting a critical role for this enzyme in gastrulation. Identification of beta-CD in an accessible model organism will create new opportunities to study vitamin A metabolism.

Amino Acid Sequence↗
PubMed2000

The upstream region of the Rpe65 gene confers retinal pigment epithelium-specific expression in vivo and in vitro and contains critical octamer and E-box binding sites.

RPE65 is essential for all-trans- to 11-cis-retinoid isomerization, the hallmark reaction of the retinal pigment epithelium (RPE). Here, we identify regulatory elements in the Rpe65 gene and demonstrate their functional relevance to Rpe65 gene expression. We show that the 5' flanking region of the mouse Rpe65 gene, like the human gene, lacks a canonical TATA box and consensus GC and CAAT boxes. The mouse and human genes do share several cis-acting elements, including an octamer, a nuclear factor one (NFI) site, and two E-box sites, suggesting a conserved mode of regulation. A mouse Rpe65 promoter/beta-galactosidase transgene containing bases -655 to +52 (TR4) of the mouse 5' flanking region was sufficient to direct high RPE-specific expression in transgenic mice, whereas shorter fragments (-297 to +52 or -188 to +52) generated only background activity. Furthermore, transient transfection of analogous TR4/luciferase constructs also directed high reporter activity in the human RPE cell line D407 but weak activity in the non-RPE cell lines HeLa, HepG2, and HS27. Functional binding of potential transcription factors to the octamer sequence, AP-4, and NFI sites was demonstrated by directed mutagenesis, electrophoretic mobility shift assay, and cross-linking. Mutations of these sites abolished binding and corresponding transcriptional activity and indicated that octamer and E-box transcription factors synergistically regulate the RPE65 promoter function. Thus, we have identified the regulatory region in the Rpe65 gene that accounts for tissue-specific expression in the RPE and found that octamer and E-box transcription factors play a critical role in the transcriptional regulation of the Rpe65 gene.

5' Untranslated Regions↗
PubMed2000

Transgenic expression of an immunologically privileged retinal antigen extraocularly enhances self tolerance and abrogates susceptibility to autoimmune uveitis.

Interphotoreceptor retinoid-binding protein (IRBP) is an immunologically privileged retinal antigen that can elicit experimental autoimmune uveitis (EAU). The nature and extent of tolerance to immunologically privileged self antigens is poorly understood. To investigate whether transgenic expression of IRBP extraocularly enhances tolerance and protects from EAU we prepared mice that express half of the mouse IRBP gene, containing a potent uveitogenic epitope (residues 161 - 180), under control of MHC class II promoter. Transgene mRNA was detectable in many tissues. Transgenic protein was undetectable by conventional assays, but was detected in thymic tissue by lymphocyte proliferation assay after induction of the promoter. Transgenic mice challenged with p161 - 180 did not develop EAU and had reduced immunological responses, but remained susceptible to EAU induced by whole IRBP, that contains additional uveitogenic epitopes. Disease was also induced by wild type T cells specific to p161 - 180. Thus, extraocular expression of a privileged retinal antigen enhances self tolerance, supporting the notion that sequestration contributes to immune privilege. Exceedingly low levels of transgene expression result in tolerance that is both profound and epitope specific, implying anergy or deletion of the endogenous uveitogenic repertoire. The same level of expression is, however, insufficient to tolerize wild-type effector T cells in the periphery.

Amino Acid Sequence↗
PubMed2000

A QTL on distal chromosome 3 that influences the severity of light-induced damage to mouse photoreceptors.

C57BL/6J-c(2J) (c2J) albino mice showed much less damage to their photoreceptors after exposure to prolonged light than BALB/c mice and seven other albino strains tested. There were no gender differences, and preliminary studies suggested that the c2J relative protective effect was a complex trait. A genome-wide scan using dinucleotide repeat markers was carried out for the analysis of 194 progeny of the backcross (c2J x BALB/c)F(1) x c2J and the thickness of the outer nuclear layer (ONL) of the retina was the quantitative trait reflecting retinal damage. Our results revealed a strong and highly significant quantitative trait locus (QTL) on mouse Chromosome (Chr) 3 that contributes almost 50% of the c2J protective effect, and three other very weak but significant QTLs on Chrs 9, 12, and 14. Interestingly, the Chrs 9 and 12 QTLs corresponded to relative susceptibility alleles in c2J (or relative protection alleles in BALB/c), the opposite of the relative protective effect of the QTLs on Chrs 3 and 14. We mapped the Rpe65 gene to the apex of the Chr 3 QTL (LOD score = 19.3). Northern analysis showed no difference in retinal expression of Rpe65 message between c2J and BALB/c mice. However, sequencing of the Rpe65 message revealed a single base change in codon 450, predicting a methionine in c2J and a leucine in BALB/c. When the retinas of aging BALB/c and c2J mice reared in normal cyclic light were compared, the BALB/c retinas showed a small but significant loss of photoreceptor cells, while the c2J retinas did not. Finding light damage-modifying genes in the mouse may open avenues of study for understanding age-related macular degeneration and other retinal degenerations, since light exposures may contribute to the course of these diseases.

Aging↗
PubMed2000

Protection of Rpe65-deficient mice identifies rhodopsin as a mediator of light-induced retinal degeneration.

Light-induced apoptosis of photoreceptors represents an animal model for retinal degeneration. Major human diseases that affect vision, such as age-related macular degeneration (AMD) and some forms of retinitis pigmentosa (RP), may be promoted by light. The receptor mediating light damage, however, has not yet been conclusively identified; candidate molecules include prostaglandin synthase, cytochrome oxidase, rhodopsin, and opsins of the cones and the retinal pigment epithelium (PE). We exposed to bright light two groups of genetically altered mice that lack the visual pigment rhodopsin (Rpe65-/- and Rho-/-). The gene Rpe65 is specifically expressed in the PE and essential for the re-isomerization of all-trans retinol in the visual cycle and thus for the regeneration of rhodopsin after bleaching. Rho-/- mice do not express the apoprotein opsin in photoreceptors, which, consequently, do not contain rhodopsin. We show that photoreceptors lacking rhodopsin in these mice are completely protected against light-induced apoptosis. The transcription factor AP-1, a central element in the apoptotic response to light, is not activated in the absence of rhodopsin, indicating that rhodopsin is essential for the generation or transduction of the intracellular death signal induced by light.

Animals↗
PubMed1999

What can we learn about age-related macular degeneration from other retinal diseases?

Age-related macular degeneration (AMD) is increasingly recognized as a complex genetic disorder in which one or more genes contribute to an individual's susceptibility for developing the condition. Twin and family studies as well as population-based genetic epidemiologic methods have convincingly demonstrated the importance of genetics in AMD, though the extent of heritability, the number of genes involved, and the phenotypic and genetic heterogeneity of the condition remain unresolved. The extent to which other hereditary macular dystrophies such as Stargardts disease, familial radial drusen (malattia leventinese), Best's disease, and peripherin/RDS-related dystrophy are related to AMD remains unclear. Alzheimer's disease, another late onset, heterogeneous degenerative disorder of the central nervous system, offers a valuable model for identifying the issues that confront AMD genetics.

ATP-Binding Cassette Transporters↗
PubMed1999

Identification of RPE65 in transformed kidney cells.

The protein RPE65 has an important role in retinoid processing and/or retinoid transport in the eye. Retinoids are involved in cell differentiation, embryogenesis and carcinogenesis. Since the kidney is known as an important site for retinoid metabolism, the expression of RPE65 in normal kidney and transformed kidney cells has been examined. The RPE65 mRNA was detected in transformed kidney cell lines including the human embryonic kidney cell line HEK293 and the African green monkey kidney cell lines COS-1 and COS-7 by reverse transcription PCR. In contrast, it was not detected in human primary kidney cells or monkey kidney tissues under the same PCR conditions. The RPE65 protein was also identified in COS-7 and HEK293 cells by Western blot analysis using a monoclonal antibody to RPE65, but not in the primary kidney cells or kidney tissues. The RPE65 cDNA containing the full-length encoding region was amplified from HEK293 and COS-7 cells. DNA sequencing showed that the RPE65 cDNA from HEK293 cells is identical to the RPE65 cDNA from the human retinal pigment epithelium. The RPE65 from COS-7 cells shares 98 and 99% sequence identity with human RPE65 at the nucleotide and amino acid levels, respectively. Moreover, the RPE65 mRNA was detected in three out of four renal tumor cultures analyzed including congenital mesoblastic nephroma and clear cell sarcoma of the kidney. These results demonstrated that transformed kidney cells express this retinoid processing protein, suggesting that these transformed cells may have an alternative retinoid metabolism not present in normal kidney cells.

Amino Acid Sequence↗
PubMed1998

Cloning and localization of RPE65 mRNA in salamander cone photoreceptor cells1.

RPE65 is a potential retinoid-processing protein expressed in the retinal pigment epithelium. Mutations in the RPE65 gene have been shown to cause certain inherited retinal dystrophies. Previous studies have shown that salamander cone photoreceptor cells have a unique retinoid processing mechanism which is distinct from that of rods. To determine whether RPE65 is expressed in photoreceptors, the RPE65 cDNA was cloned from a salamander retinal cDNA library. The deduced protein consists of 533 amino acids and is 85% identical to human and bovine RPE65. The RPE65 mRNA was detected in all of the single cone cells isolated from the salamander retina, as well as in the retinal pigment epithelium by RT-PCR, but not in the isolated rods. The RT-PCR products have been confirmed to be RPE65 by DNA sequencing. The results indicate that this potential retinoid processing protein is expressed in the cone photoreceptor cells but not in rods. Therefore, this protein may contribute to the unique retinoid processing capabilities in salamander cones.

Amino Acid Sequence↗
PubMed1998

Role of the 3'-untranslated region of RPE65 mRNA in the translational regulation of the RPE65 gene: identification of a specific translation inhibitory element.

Previously, we demonstrated that explanted bovine retinal pigment epithelium (RPE) cells lose RPE65 protein, a major microsomal protein specific to RPE, while the RPE65 mRNA remains, suggesting posttranscriptional regulation of RPE65 expression in vitro. Accordingly, we analyze here the effect of the 5'- and 3'-untranslated regions (UTRs) of RPE65 mRNA on translational efficiency using in vitro translation systems. We compared the levels of translation products and mRNA stability among RPE65 transcripts containing deletions of the 5'- and 3'-UTRs. First, the 5'-UTR does not affect translational efficiency. However, the 3'-UTR does influence translation efficiency. A putative translation inhibitory element (TIE) is contained within the 170-nucleotide (nt) sequence downstream of the stop codon. There is also a weak destabilizing effect that is associated with the region 3' to the putative TIE. But the effect of this is much less than that of the TIE. This TIE, however, does not inhibit translation of the heterologous chloramphenicol acetyltransferase gene, suggesting that a specific interaction with the upstream RPE65 coding sequence, or its product, may be required. Thus, the posttranscriptional regulation of RPE65 mRNA expression observed in cultured RPE may be via a mechanism of translational inhibition.

Animals↗
PubMed1998

Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle.

Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65-/- mice are disorganized compared with those of Rpe65+/+ and Rpe65+/- mice. Rod function, as measured by electroretinography, is abolished in Rpe65-/- mice, although cone function remains. Rpe65-/- mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65-/- mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.

Animals↗
PubMed1997

RPE65, the major retinal pigment epithelium microsomal membrane protein, associates with phospholipid liposomes.

The retinal pigment epithelium (RPE)-specific protein RPE65 is the major protein of the RPE microsomal membrane fraction. Though RPE65 lacks transmembrane domains or signal peptide, detergents are required for its maximally effective solubilization in isotonic buffers. However, in 0.75-1.0 M KCl, RPE65 is as soluble without detergent, indicating a peripheral membrane association. We wished to understand why this non-membrane-inserted protein was so closely associated with RPE microsomal membranes. To explore the possible involvement of interactions with phospholipids, an isotonic salt-soluble extract of RPE was incubated with phosphatidylcholine (PC)/phosphatidylserine (PS)/phosphatidylinositol liposomes and centrifuged to sediment the liposomes. RPE65 cosedimented with the liposome pellet. RPE65 also cosedimented with synthetic dipalmitoyl-, 1-palmitoyl, 2-docosahexaenoyl-PC or dipalmitoyl-PS liposomes. Incubation with 1 mM Ca2+ or 1 mM EGTA had no effect, indicating a Ca2+-independent association. A spectrophotometric assay showed that this interaction of RPE65 with phospholipid vesicles resulted in increased light scattering, consistent with phospholipid vesicle aggregation. Resonance energy transfer experiments showed that any putative aggregation occurred without subsequent vesicle fusion. This PC affinity was further confirmed by incubation of RPE extract with dimyristoyl-PC-immobilized artificial membrane (IAM.PC) matrix. The RPE65 selectively bound and was elutable with 2% detergent. This RPE65-phospholipid liposome association may explain the solubilization characteristics of RPE65 and may be related to the function of RPE65 and to its physical association with the RPE smooth endoplasmic reticulum.

Animals↗