[Show abstract][Hide abstract] ABSTRACT: Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is required for the biosynthesis of photoreceptor phosphodiesterase (PDE). Gene defects in AIPL1 cause a heterogeneous set of conditions ranging from Leber's congenital amaurosis (LCA), the severest form of early-onset retinal degeneration, to milder forms such as retinitis pigmentosa (RP) and cone-rod dystrophy. In mice, null and hypomorphic alleles cause retinal degeneration similar to human LCA and RP, respectively. Thus these mouse models represent two ends of the disease spectrum associated with AIPL1 gene defects in humans. We evaluated whether adeno-associated virus (AAV)-mediated gene replacement therapy in these models could restore PDE biosynthesis in rods and cones and thereby improve photoreceptor survival. We validated the efficacy of human AIPL1 (isoform 1) replacement gene controlled by a promoter derived from the human rhodopsin kinase (RK) gene, which is active in both rods and cones. We found substantial and long-term rescue of the disease phenotype as a result of transgene expression. This is the first gene therapy study in which both rods and cones were targeted successfully with a single photoreceptor-specific promoter. We propose that the vector and construct design used in this study could serve as a prototype for a human clinical trial.
[Show abstract][Hide abstract] ABSTRACT: Circadian clocks are widely distributed in mammalian tissues, but little is known about the physiological functions of clocks outside the suprachiasmatic nucleus of the brain. The retina has an intrinsic circadian clock, but its importance for vision is unknown. Here, we show that mice lacking Bmal1, a gene required for clock function, had abnormal retinal transcriptional responses to light and defective inner retinal electrical responses to light, but normal photoreceptor responses to light and retinas that appeared structurally normal as observed by light and electron microscopy. We generated mice with a retina-specific genetic deletion of Bmal1, and they had defects of retinal visual physiology essentially identical to those of mice lacking Bmal1 in all tissues and lacked a circadian rhythm of inner retinal electrical responses to light. Our findings indicate that the intrinsic circadian clock of the retina regulates retinal visual processing in vivo.
Cold Spring Harbor Symposia on Quantitative Biology 02/2007; 72:307-18.
[Show abstract][Hide abstract] ABSTRACT: The actin-binding LIM domain protein (abLIM) is the mammalian homologue of UNC-115, a protein mediating axon guidance in C. elegans. AbLIM is widely expressed with three isoforms differing from one another by the length of their amino termini. Experiments utilizing dominant-negative mutants in the chick retina suggested a role for abLIM in axon path finding in retinal ganglion cells (RGCs). To investigate which variant is involved in the regulation of mammalian RGC axon guidance, we analyzed their expression profile in mice. The longest variant, abLIM-L, is highly enriched in the ganglion cell layer. AbLIM-L is up-regulated postnatally which temporally overlaps with the period of RGC axon remodeling. In contrast, the abLIM-M and abLIM-S variants are widespread and remain relatively constant through development. By selective gene targeting, we ablated abLIM-L to explore its functional significance in vivo. AbLIM-L mutant mice exhibit no apparent morphological or functional defects in photoreceptors and inner retinal neurons. Retinofugal projections and synaptic maturation also appear normal. These data suggest that abLIM-M is likely the isoform performing the essential function related to axon guidance.
[Show abstract][Hide abstract] ABSTRACT: To test the hypothesis that a lack of Tubby-like protein 1 (TULP1) function causes aberrant transport of nascent rhodopsin and to examine the functional relationship between the homologous proteins TULP1 and Tubby by studying mice carrying combined mutations.
Subcellular localization of TULP1 and rhodopsin in photoreceptors was determined by immunofluorescence and by postembedding immunoelectron microscopy. Mice carrying different tulp1/tubby allele combinations were examined by histology, electroretinograms (ERGs), and immunofluorescence microscopy.
TULP1 is distributed throughout the photoreceptor cytoplasm but is excluded from the outer segments and the nuclei. In the tulp1-/- mice, ectopic accumulation of rhodopsin occurs at an early age. Both the vesicular profiles in the interphotoreceptor space and the inner segment plasma membranes are immunoreactive for rhodopsin. Mice doubly homozygous for null mutations in the tulp1 and tubby genes initially develop photoreceptors and express a battery of photoreceptor markers at age 14 days. Thereafter their photoreceptors undergo a fulminant degeneration that reaches completion by postnatal day 17. The disease phenotype in the double homozygote is much more severe than either single homozygote. Double heterozygotes are phenotypically normal.
A lack of TULP1 function results in misrouting of nascent rhodopsin. TULP1 may be a component of the cellular machinery that targets nascent rhodopsin to the outer segments. Comparison of disease phenotypes in the single and double mutants suggests that TULP1 and Tubby are not functionally interchangeable in photoreceptors nor do they form an obligate functional complex.
[Show abstract][Hide abstract] ABSTRACT: We isolated and characterized the entire coding sequence of a human gene encoding a protein that interacts with RPGR, a protein that is absent or mutant in many cases of X-linked retinitis pigmentosa. The newly identified gene, called "RPGRIP1" for RPGR-interacting protein (MIM 605446), is located within 14q11, and it encodes a protein predicted to contain 1,259 amino acids. Previously published work showed that both proteins, RPGR and RPGRIP1, are present in the ciliary structure that connects the inner and outer segments of rod and cone photoreceptors. We surveyed 57 unrelated patients who had Leber congenital amaurosis for mutations in RPGRIP1 and found recessive mutations involving both RPGRIP1 alleles in 3 (6%) patients. The mutations all create premature termination codons and are likely to be null alleles. Patients with RPGRIP1 mutations have a degeneration of both rod and cone photoreceptors, and, early in life, they experience a severe loss of central acuity, which leads to nystagmus.
The American Journal of Human Genetics 06/2001; 68(5):1295-8. · 10.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retinitis pigmentosa (RP) is a blinding retinal disease in which the photoreceptor cells degenerate. Mutations in the gene for retinitis pigmentosa GTPase regulator (RPGR) are a frequent cause of RP. The function of RPGR is not well understood, but it is thought to be a putative guanine nucleotide exchange factor for an unknown G protein. Ablation of the RPGR gene in mice suggested a role in maintaining the polarized distribution of opsin across the cilia. To investigate its function, we used a protein interaction screen to identify candidate proteins that may interact physiologically with RPGR. One such protein, designated RPGR-interacting protein (RPGRIP), is expressed specifically in rod and cone photoreceptors. It consists of an N-terminal region predicted to form coiled coil structures linked to a C-terminal tail that binds RPGR. In vivo, both proteins co-localize in the photoreceptor connecting cilia. RPGRIP is stably associated with the ciliary axoneme independent of RPGR and is resistant to extraction under conditions that partially solubilized other cytoskeletal components. When over-expressed in heterologous cell lines, RPGRIP appears in insoluble punctate and filamentous structures. These data suggest that RPGRIP is a structural component of the ciliary axoneme, and one of its functions is to anchor RPGR within the cilium. RPGRIP is the only protein known to localize specifically in the photoreceptor connecting cilium. As such, it is a candidate gene for human photoreceptor disease. The tissue-specific expression of RPGRIP explains why mutations in the ubiquitously expressed RPGR confer a photoreceptor-specific phenotype.
Journal of Biological Chemistry 05/2001; 276(15):12091-9. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The retinal degeneration B (rdgB) gene in Drosophila is essential for photoreceptor function and survival. The rdgB mutant fly exhibits an abnormal electroretinogram and a light-dependent photoreceptor degeneration. The function of RdgB is not fully understood, but the presence of a phosphatidylinositol transfer protein domain suggests a possible role in phosphatidylinositol metabolism and signaling. Two mammalian homologs, M-RdgB1 and M-RdgB2, are known. While M-RdgB1 is widely expressed, M-RdgB2 is found primarily in the retina and the dentate gyrus. Functional conservation between the Drosophila and mammalian RdgBs was demonstrated by the ability of both M-RdgBs to rescue the photoreceptor phenotype in rdgB mutant flies through transgenic expression. To investigate the role of M-RdgB2 in the mammalian retina, we disrupted the m-rdgB2 gene in mice by gene targeting. The homozygous knockout mice are fertile and apparently healthy. By light microscopy, immunocytochemistry and electroretinograms, mice up to 18 months of age showed normal photoreceptor function and survival. The inner retinal neurons were also examined by immunolabeling with a number of cell-specific markers and no apparent defects were found in the major cell populations. We conclude that M-rdgB2 is not essential for phototransduction and photoreceptor survival. Thus, m-rdgB2 is not a candidate gene for human retinal degenerations. Whether M-rdgB2 has a role in visual processing in the inner retina, or whether it is required for hippocampal function, remains to be determined.
[Show abstract][Hide abstract] ABSTRACT: The X-linked RP3 locus codes for retinitis pigmentosa GTPase regulator (RPGR), a protein of unknown function with sequence homology to the guanine nucleotide exchange factor for Ran GTPase. We created an RPGR-deficient murine model by gene knockout. In the mutant mice, cone photoreceptors exhibit ectopic localization of cone opsins in the cell body and synapses and rod photoreceptors have a reduced level of rhodopsin. Subsequently, both cone and rod photoreceptors degenerate. RPGR was found normally localized to the connecting cilia of rod and cone photoreceptors. These data point to a role for RPGR in maintaining the polarized protein distribution across the connecting cilium by facilitating directional transport or restricting redistribution. The function of RPGR is essential for the long-term maintenance of photoreceptor viability.
Proceedings of the National Academy of Sciences 04/2000; 97(7):3649-54. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Tulp1 gene is a member of the tubby gene family with unknown function. Mutations in the human TULP1 gene cause autosomal recessive retinitis pigmentosa. To understand the pathogenic mechanism associated with TULP1 mutations and to explore the physiologic function of this protein, we examined tissue distribution of the Tulp1 protein in normal mice and the photoreceptor disease phenotype in Tulp1-ablated mice.
Tissue distribution of the Tulp1 protein in normal mice was examined by immunoblotting and immunocytochemistry. The disease phenotype in tulp1-/- mice was studied by light and electron microscopy, electroretinography (ERG), and immunocytochemistry. These results were compared with another mouse model of retinal degeneration carrying a rhodopsin mutation.
Tulp1 is found exclusively in photoreceptors, localizing predominantly in the inner segments. It is a soluble protein with an apparent molecular weight of approximately 70 kDa. Photoreceptor degeneration developed in tulp1-/- mice, with early involvement of both rods and cones. At the early stage of degeneration, rod and cone opsins, but not peripherin/RDS, exhibited prominent ectopic localization. Electron microscopy revealed massive accumulation of extracellular vesicles surrounding the distal inner segments.
The function of Tulp1 is required to maintain viability of rod and cone photoreceptors. Extracellular vesicular accumulation is not a common phenomenon associated with photoreceptor degeneration but appears to be a distinct ultrastructural feature shared by a small group of retinal disease models. The defect in tulp1-/- mice may be consistent with a loss of polarized transport of nascent opsin to the outer segments.
[Show abstract][Hide abstract] ABSTRACT: Mutations in the Drosophila retinal degeneration B (rdgB) gene cause a rapid loss of the electrophysiological light response and subsequent light-enhanced photoreceptor degeneration. The rdgB gene encodes a protein with an N-terminal phosphatidylinositol transfer protein domain, a large C-terminal segment, and several hydrophobic regions thought to multiply span the subrhabdomeric cisternal membrane. A mammalian rdgB homolog (m-rdgB1) was previously identified and shown to exhibit widespread tissue distribution and functionally rescue the Drosophila rdgB mutant phenotypes. We describe a second mammalian rdgB homolog (m-rdgB2) that possesses 46% amino acid identity to Drosophila RdgB and 56% identity to M-RdgB1. M-RdgB2 possesses a neuronal-specific expression pattern, with high levels in the retina and the dentate gyrus mossy fibers and dendritic field. Using M-RdgB2-specific antibodies and subcellular fractionation, we demonstrate that M-RdgB2 is not an integral membrane protein but is stably associated with a particulate fraction through protein-protein interactions. Although transgenic expression of M-RdgB2 in rdgB2 null mutant flies suppressed the retinal degeneration, it failed to fully restore the electrophysiological light response. Because transgenic expression of M-RdgB2 does not restore the wild-type phenotype to rdgB2 mutant flies to the same extent as M-RdgB1, functional differences likely exist between the two M-RdgB homologs.
Journal of Neuroscience 10/1999; 19(17):7317-25. · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A therapeutic effect of vitamin A supplementation on the course of photoreceptor degeneration, previously reported for patients with retinitis pigmentosa, was tested in two transgenic mouse models of this disease, each carrying a dominant rhodopsin mutation. The threonine-17 --> methionine (T17M) mutation is a class II rhodopsin mutation, characterized by a thermal instability/folding defect and minimal regeneration with the chromophore. The proline-347 --> serine (P347S) mutation belongs to class I, comprised of a smaller number of mutations that exhibit no recognized biochemical abnormality in vitro. In the present study, each of the two mouse models was fed a diet containing 2.5 mg of vitamin A palmitate (control) or 102.5 mg of vitamin A palmitate (high vitamin A) per kilogram of diet. Dark-adapted, full-field electroretinograms showed that the high vitamin A diet significantly reduced the rate of decline of a-wave and b-wave amplitudes in the T17M mice but had no significant effect on the decline of electroretinogram amplitude in the P347S mice. Correspondingly, histologic evaluation revealed that the treatment was associated with significantly longer photoreceptor inner and outer segments and a thicker outer nuclear layer in the T17M mice but had no effect on photoreceptor morphology in the P347S mice. In a separate series of experiments, the instability defect of the T17M mutant opsin expressed in vitro was partially alleviated by inclusion of 11-cis-retinal in the culture media. These results show that vitamin A supplementation slows the rate of photoreceptor degeneration caused by a class II rhodopsin mutation. Vitamin A supplementation may confer therapeutic benefit by stabilizing mutant opsins through increased availability of the chromophore.
Proceedings of the National Academy of Sciences 10/1998; 95(20):11933-8. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecules that couple the actin-based cytoskeleton to intracellular signaling pathways are central to the processes of cellular morphogenesis and differentiation. We have characterized a novel protein, the actin-binding LIM (abLIM) protein, which could mediate such interactions between actin filaments and cytoplasmic targets. abLIM protein consists of a COOH-terminal cytoskeletal domain that is fused to an NH2-terminal domain consisting of four double zinc finger motifs. The cytoskeletal domain is approximately 50% identical to erythrocyte dematin, an actin-bundling protein of the red cell membrane skeleton, while the zinc finger domains conform to the LIM motif consensus sequence. In vitro expression studies demonstrate that abLIM protein can bind to F-actin through the dematin-like domain. Transcripts corresponding to three distinct isoforms have a widespread tissue distribution. However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments. Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners. The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments.
The Journal of Cell Biology 09/1997; 138(3):575-88. · 9.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To explore the pathogenic mechanism of dominant mutations affecting the carboxyl terminus of rhodopsin that cause retinitis pigmentosa, we generated five lines of transgenic mice carrying the proline-347 to serine (P347S) mutation. The severity of photoreceptor degeneration correlated with the levels of transgene expression in these lines. Visual function as measured by the electroretinogram was approximately normal at an early age when there was little histologic evidence of photoreceptor degeneration, but it deteriorated as photoreceptors degenerated. Immunocytochemical staining showed the mutant rhodopsin predominantly in the outer segments prior to histologically evident degeneration, a finding supported by quantitation of signal intensities in different regions of the photoreceptor cells by confocal microscopy. A distinct histopathologic abnormality was the accumulation of submicrometer-sized vesicles extracellularly near the junction between inner and outer segments. The extracellular vesicles were bound by a single membrane that apparently contained rhodopsin as revealed by ultrastructural immunocytochemical staining with anti-rhodopsin antibodies. The outer segments, although shortened, contained well-packed discs. Proliferation of the endoplasmic reticulum as reported in Drosophila expressing dominant rhodopsin mutations was not observed. The accumulation of rhodopsinladen vesicles likely represents aberrant transport of rhodopsin from the inner segments to the nascent disc membranes of the outer segments. It is possible that photoreceptor degeneration occurs because of a failure to renew outer segments at a normal rate, thereby leading to a progressive shortening of outer segments, or because of the loss of cellular contents to the extracellular space, or because of both.
Proceedings of the National Academy of Sciences 12/1996; 93(24):14176-81. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have studied the use of adenovirus-mediated gene transfer to reverse the pathologic changes of lysosomal storage disease caused by beta-glucuronidase deficiency in the eyes of mice with mucopolysaccharidosis VII. A recombinant adenovirus carrying the human beta-glucuronidase cDNA coding region under the control of a non-tissue-specific promoter was injected intravitreally or subretinally into the eyes of mice with mucopolysaccharidosis VII. At 1-3 weeks after injection, the treated and control eyes were examined histochemically for beta-glucuronidase expression and histologically for phenotypic correction of the lysosomal storage defect. Enzymatic expression was detected 1-3 weeks after injection. Storage vacuoles in the retinal pigment epithelium (RPE) were still present 1 week after gene transfer but were reduced to undetectable levels by 3 weeks in both intravitreally and subretinally injected eyes. There was minimal evidence of ocular pathology associated with the viral injection. These data indicate that adenovirus-mediated gene transfer to the eye may provide for adjunctive therapy for lysosomal storage diseases affecting the RPE in conjunction with enzyme replacement and/or gene therapies for correction of systemic disease manifestations. The data also support the view that recombinant adenovirus may be useful as a gene therapy vector for retinal degenerations that result from a primary genetic defect in the RPE cells.
Proceedings of the National Academy of Sciences 09/1995; 92(17):7700-4. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The missense mutation Lys-296-->Glu (K296E) in the rhodopsin gene produces an opsin with no chromophore binding site and therefore is not activated by light. Nevertheless, the mutant opsin constitutively activates transducin in vitro and causes photoreceptor degeneration in vivo, possibly by continuously activating the phototransduction cascade, analogous to constant exposure to environmental light. We studied the K296E mutation in eight lines of transgenic mice. Each line developed photoreceptor degeneration with the rate of degeneration increasing monotonically as the ratio of mutant:wild-type opsin mRNA increased. At no time in the course of degeneration was there endogenous light adaptation in the retina as measured by the electroretinogram. The mutant opsin was found to be invariably phosphorylated and stably bound to arrestin. Light-independent activation of transducin was demonstrated only after the removal of arrestin and dephosphorylation of K296E opsin. Thus, K296E opsin in vivo does not activate the phototransduction cascade because it is shut off by photoreceptor inactivation mechanisms. Our data show that the K296E mutation does not cause photoreceptor degeneration by continuous activation of phototransduction.
Proceedings of the National Academy of Sciences 05/1995; 92(8):3551-5. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hereditary degenerations and dysfunctions of the retina are an extremely heterogeneous group of diseases. This summary deals with recent advances in the molecular genetics of a subset of those disorders, namely, those encompassed under the diagnosis 'retinitis pigmentosa'. Over 20 loci where mutations cause retinitis pigmentosa have been mapped; the review focuses on the seven retinitis pigmentosa loci that have been identified.
[Show abstract][Hide abstract] ABSTRACT: The ability of replication-deficient adenovirus to mediate gene transfer to retinal cells was evaluated.
A replication-deficient adenoviral vector, AdCMV beta A.ntlacZ, which contains the bacterial beta-galactosidase (lacZ) reporter gene, was injected into the subretinal space of normal, rd, and rds strains of mice at various ages. The efficiency and duration of transgene expression were assessed by histochemical examination and transmission electron microscopy.
AdCMV beta A.ntlacZ was effective in mediating gene transfer to the retinal pigment epithelial cells, rod and cone photoreceptor cells, and cells in the inner nuclear layer of the retina for periods of up to 1 month. Gene transfer to retinal pigment epithelial cells occurred at much lower viral titers than was required for gene transfer to photoreceptor cells. The extent to which photoreceptor cells could be transduced varied with the age of the animals and the conditions of the photoreceptor cells: greater numbers of photoreceptor cells were transduced in 5- to 7-day-old pups and in mice at the initial stages of photoreceptor degeneration than in normal adult mice. No evidence of gross pathogenic effects or viremia in recipient mice was observed.
Replication-deficient adenovirus mediates transfer and expression of a foreign gene in retinal pigment epithelial and photoreceptor cells. Gene transfer to photoreceptor cells is enhanced in developing retinas or at the predegenerate stage of photoreceptors in genetically programmed retinal degeneration.