Cone Degeneration Research Articles

CNTF Induces Regeneration of Cone Outer Segments in a Rat Model of Retinal Degeneration

BackgroundCone photoreceptors are responsible for color and central vision. In the late stage of retinitis pigmentosa and in geographic atrophy associated with age-related macular degeneration, cone degeneration eventually causes loss of central vision. In the present work, we investigated cone degeneration secondary to rod loss in the S334ter-3 transgenic rats carrying the rhodopsin mutation S334ter.Methodology/Principal FindingsRecombinant human ciliary neurotrophic factor (CNTF) was delivered by intravitreal injection to the left eye of an animal, and vehicle to the right eye. Eyes were harvested 10 days after injection. Cone outer segments (COS), and cell bodies were identified by staining with peanut agglutinin and cone arrestin antibodies in whole-mount retinas. For long-term treatment with CNTF, CNTF secreting microdevices were implanted into the left eyes at postnatal day (PD) 20 and control devices into the right eyes. Cone ERG was recorded at PD 160 from implanted animals. Our results demonstrate that an early sign of cone degeneration is the loss of COS, which concentrated in many small areas throughout the retina and is progressive with age. Treatment with CNTF induces regeneration of COS and thus reverses the degeneration process in early stages of cone degeneration. Sustained delivery of CNTF prevents cones from degeneration and helps them to maintain COS and light-sensing function.Conclusions/SignificanceLoss of COS is an early sign of secondary cone degeneration whereas cell death occurs much later. At early stages, degenerating cones are capable of regenerating outer segments, indicating the reversal of the degenerative process. Sustained delivery of CNTF preserves cone cells and their function. Long-term treatment with CNTF starting at early stages of degeneration could be a viable strategy for preservation of central vision for patients with retinal degenerations.

Autosomal Recessive Retinitis Pigmentosa with Early Macular Affectation Caused by Premature Truncation inPROM1

To identify the genetic basis of a large consanguineous Spanish pedigree affected with autosomal recessive retinitis pigmentosa (arRP) with premature macular atrophy and myopia. After a high-throughput cosegregation gene chip was used to exclude all known RP and Leber congenital amaurosis (LCA) candidates, genome-wide screening and linkage analysis were performed. Direct mutational screening identified the pathogenic mutation, and primers were designed to obtain the RT-PCR products for isoform characterization. Mutational analysis detected a novel homozygous PROM1 mutation, c.869delG in exon 8 cosegregating with the disease. This variant causes a frameshift that introduces a premature stop codon, producing truncation of approximately two-thirds of the protein. Analysis of PROM1 expression in the lymphocytes of patients, carriers, and control subjects revealed an aberrant transcript that is degraded by the nonsense-mediated decay pathway, suggesting that the disease is caused by the absence of the PROM1 protein. Three (s2, s11 and s12) of the seven alternatively spliced isoforms reported in humans, accounted for 98% of the transcripts in the retina. Given that these three contained exon 8, no PROM1 isoform is expected in the affected retinas. A remarkable clinical finding in the affected family is early macular atrophy with concentric spared areas. The authors propose that the hallmark of PROM1 truncating mutations is early and severe progressive degeneration of both rods and cones and highlight this gene as a candidate of choice to prioritize in the molecular genetic study of patients with noncanonical clinical peripheral and macular affectation.

The Leber congenital amaurosis protein, AIPL1, is needed for the viability and functioning of cone photoreceptor cells

Leber congenital amaurosis (LCA) caused by mutations in Aryl hydrocarbon receptor interacting protein like-1 (Aipl1) is a severe form of childhood blindness. At 4 weeks of age, a mouse model of LCA lacking AIPL1 exhibits complete degeneration of both rod and cone photoreceptors. Rod cell death occurs due to rapid destabilization of rod phosphodiesterase, an enzyme essential for rod survival and function. However, little is understood regarding the role of AIPL1 in cone photoreceptors. Cone degeneration observed in the absence of AIPL1 could be due to an indirect 'bystander effect' caused by rod photoreceptor death or a direct role for AIPL1 in cones. To understand the importance of AIPL1 in cone photoreceptor cells, we transgenically expressed hAIPL1 exclusively in the rod photoreceptors of the Aipl1(-/-) mouse. Transgenic expression of hAIPL1 restored rod morphology and the rod-derived electroretinogram response, but cone photoreceptors were non-functional in the absence of AIPL1. In addition, the cone photoreceptors degenerate, but at a slower rate compared with Aipl1(-/-) mice. This degeneration is linked to the highly reduced levels of cone PDE6 observed in the hAIPL1 transgenic mice. Our studies demonstrate that AIPL1 is needed for the proper functioning and survival of cone photoreceptors. However, rod photoreceptors also provide support that partially preserves cone photoreceptors from rapid death in the absence of AIPL1.

Trafficking of Membrane Proteins to Cone But Not Rod Outer Segments Is Dependent on Heterotrimeric Kinesin-II

Heterotrimeric kinesin-II is a molecular motor localized to the inner segment, connecting cilium and axoneme of mammalian photoreceptors. Our purpose was to identify the role of kinesin-II in anterograde intraflagellar transport by photoreceptor-specific deletions of kinesin family member 3A (KIF3A), its obligatory motor subunit. In cones lacking KIF3A, membrane proteins involved in phototransduction did not traffic to the outer segments resulting in complete absence of a photopic electroretinogram and progressive cone degeneration. Rod photoreceptors lacking KIF3A degenerated rapidly between 2 and 4 weeks postnatally, but the phototransduction components including rhodopsin trafficked to the outer segments during the course of degeneration. Furthermore, KIF3A deletion did not affect synaptic anterograde trafficking. The results indicate that trafficking of membrane proteins to the outer segment is dependent on kinesin-II in cone, but not rod photoreceptors, even though rods and cones share similar structures, and closely related phototransduction polypeptides.

The homeobox gene CHX10/VSX2 regulates RdCVF promoter activity in the inner retina

Rod-derived Cone Viability Factor (RdCVF) is a trophic factor with therapeutic potential for the treatment of retinitis pigmentosa, a retinal disease that commonly results in blindness. RdCVF is encoded by Nucleoredoxin-like 1 (Nxnl1), a gene homologous with the family of thioredoxins that participate in the defense against oxidative stress. RdCVF expression is lost after rod degeneration in the first phase of retinitis pigmentosa, and this loss has been implicated in the more clinically significant secondary cone degeneration that often occurs. Here, we describe a study of the Nxnl1 promoter using an approach that combines promoter and transcriptomic analysis. By transfection of selected candidate transcription factors, chosen based upon their expression pattern, we identified the homeodomain proteins CHX10/VSX2, VSX1 and PAX4, as well as the zinc finger protein SP3, as factors that can stimulate both the mouse and human Nxnl1 promoter. In addition, CHX10/VSX2 binds to the Nxnl1 promoter in vivo. Since CHX10/VSX2 is expressed predominantly in the inner retina, this finding motivated us to demonstrate that RdCVF is expressed in the inner as well as the outer retina. Interestingly, the loss of rods in the rd1 mouse, a model of retinitis pigmentosa, is associated with decreased expression of RdCVF by inner retinal cells as well as by rods. Based upon these results, we propose an alternative therapeutic strategy aimed at recapitulating RdCVF expression in the inner retina, where cell loss is not significant, to prevent secondary cone death and central vision loss in patients suffering from retinitis pigmentosa.

Gene delivery to mitotic and postmitotic photoreceptors Via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa

The purpose of the present study was to test the therapeutic efficiency and safety of compacted-DNA nanoparticle-mediated gene delivery into the subretinal space of a juvenile mouse model of retinitis pigmentosa. Nanoparticles containing the mouse opsin promoter and wild-type mouse Rds gene were injected subretinally into mice carrying a haploinsufficiency mutation in the retinal degeneration slow (rds(+ or -)) gene at postnatal day (P)5 and 22. Control mice were either injected with saline, injected with uncompacted naked plasmid DNA carrying the Rds gene, or remained untreated. Rds mRNA levels peaked at postinjection day 2 to 7 (PI-2 to PI-7) for P5 injections, stabilized at levels 2-fold higher than in uninjected controls for both P5 and P22 injections, and remained elevated at the latest time point examined (PI-120). Rod function (measured by electroretinography) showed modest but statistically significant improvement compared with controls after both P5 and P22 injections. Cone function in nanoparticle-injected eyes reached wild-type levels for both ages of injections, indicating full prevention of cone degeneration. Ultrastructural examination at PI-120 revealed significant improvement in outer segment structures in P5 nanoparticle-injected eyes, while P22 injection had a modest structural improvement. There was no evidence of macrophage activation or induction of IL-6 or TNF-alpha mRNA in P5 or P22 nanoparticle-dosed eyes at either PI-2 or PI-30. Thus, compacted-DNA nanoparticles can efficiently and safely drive gene expression in both mitotic and postmitotic photoreceptors and retard degeneration in this model. These findings, using a clinically relevant treatment paradigm, illustrate the potential for application of nanoparticle-based gene replacement therapy for treatment of human retinal degenerations.-Cai, X., Conley, S. M., Nash, Z., Fliesler, S. J., Cooper, M. J., Naash, M. I. Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa.

Impaired cone function and cone degeneration resulting from CNGB3 deficiency: down-regulation of CNGA3 biosynthesis as a potential mechanism

The cone cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. This channel is composed of two structurally related subunits, CNGA3 and CNGB3; CNGA3 is the ion-conducting subunit, whereas CNGB3 is a modulatory subunit. Mutations in both subunits are associated with achromatopsia and progressive cone dystrophy, with mutations in CNGB3 alone accounting for 50% of all known cases of achromatopsia. However, the molecular mechanisms underlying cone diseases that result from CNGB3 deficiency are unknown. This study investigated the role of CNGB3 in cones, using CNGB3(-/-) mice. Cone dysfunction was apparent at the earliest time point examined (post-natal day 30) in CNGB3(-/-) mice. When compared with wild-type (WT) controls: photopic electroretingraphic (ERG) responses were decreased by approximately 75%, whereas scotopic ERG responses were unchanged; visual acuity was decreased by approximately 20%, whereas contrast sensitivity was unchanged; cone density was reduced by approximately 40%; photoreceptor apoptosis was detected; and outer segment disorganization was observed in some cones. Notably, CNGA3 protein and mRNA levels were significantly decreased in CNGB3(-/-) mice; in contrast, mRNA levels of S-opsin, Gnat2 and Pde6c were unchanged, relative to WT mice. Hence, we show that loss of CNGB3 reduces biosynthesis of CNGA3 and impairs cone CNG channel function. We suggest that down-regulation of CNGA3 contributes to the pathogenic mechanism by which CNGB3 mutations lead to human cone disease.

In Vivo Analysis of Cone Survival in Mice

To identify individual cone photoreceptors in a transgenic mouse line in vivo based on selective expression of green fluorescent protein (GFP) using cSLO (confocal scanning laser ophthalmoscopy) and to use this approach to monitor cone cell fate in mouse models of retinal degeneration. Transgenic mice expressing GFP under the control of a red-green opsin promoter (RG-GFP mice) were analyzed in vivo with respect to GFP expression in cone cells using cSLO and functional integrity using electroretinography (ERG). Histology was performed to correlate the pattern of GFP expression with light microscopic data. Longitudinal monitoring of cone survival was evaluated in crossbreds of RG-GFP mice with cpfl1 and Rpe65(-/-) mutant mice, respectively. The authors found that RG-GFP transgenic mice had a stable GFP expression that did not interfere with retinal function up to at least 3 months of age. Thus, a longitudinal analysis of cone degeneration in individual RG cpfl1 and RG Rpe65(-/-) cross-bred mice in vivo was successfully performed and demonstrated distinct time frames of cone survival in the particular mouse model. Monitoring GFP expression in cone photoreceptor cells, such as in the RG-GFP mouse, is a promising in vivo approach for the analysis of cone survival in mice.

Gene therapy mediates cone rescue and rejuvenation in the R91W mutant form of Rpe65‐deficiency mice

Abstract Purpose Given the advances of gene therapy studies to cure RPE65‐derived Leber Congenital Amaurosis (LCA) (clinical trials phase I), it is of prime importance to examine how cones can be rescued in different mutant contexts. Consequently, we evaluated the effect on retinal activity and cone survival of lentivirus‐mediated gene therapy in the R91W knock‐in mouse model expressing the mutant Rpe65R91W gene. Methods An HIV‐1‐derived lentiviral vector (LV) expressing either the GFP or the mouse Rpe65 cDNA under the control of a 0.8 kb fragment of the human Rpe65 promoter (R0.8) was produced. LV‐R0.8‐RPE65 or GFP was injected into 5‐days‐old (P5) or 1 month‐old R91W mice. Functional and morphological retinal rescues were investigated at 4 months of age. Results Increased light sensitivity was detected by ERG and pupillary light responses in animals injected with LV‐R0.8‐RPE65 at both P5 and 1 month compared to controls. Histological analysis showed improved expression of cone markers and cone outersegment morphology. Furthermore, the density of cones in the region of RPE65 delivery after treatment at P5 reached the wild type level. However, before injection at 1 month of age, only a fraction of the cones (40% of the number found in WT animals) in the Rpe65R91W/R91W mice expressed cone transducin, this fraction increased to 64% after treatment. Moreover, these cones appeared normal. Conclusion We show that lentivirus‐mediated Rpe65 gene transfer is very efficacious in early treatments and still efficient during the course of cone degeneration. Moreover, the treatment at 1 month shows a rejuvenation process of the diseased cones. Thus patient suffering from R91W mutation might benefit from a prolonged therapeutic window.

Changes in thyroid hormone reception precede SWS1 opsin downregulation in trout retina

Rainbow trout undergo natural cone degeneration and thus are interesting models for examining mechanisms of neural degeneration. They have ultraviolet-sensitive (UVS) cones that are lost over most of the retina during development; only a small functional population remains in the dorsal retina. How this spatial distribution of UVS cones is maintained is unclear. Thyroxine (T4) induces UVS cone loss, and local thyroid hormone regulation was hypothesized to control UVS cone distribution. Thyroid hormone receptor alpha (TRalpha), thyroid hormone receptor beta (TRbeta) and Type 2 deiodinase (D2) regulate thyroid hormone exposure to target cells. Regional retinal expression of these genes was investigated during exogenous T4 treatment and natural smoltification of rainbow trout. Each retina from dark-adapted parr, T4-treated parr and natural smolts was divided into four quadrants, and total RNA was isolated. Quantitative real-time RT-PCR analysis demonstrated that all retinal quadrants had increased accumulation of TRbeta transcripts 2 days post-T4 treatment, corresponding to initiation of SWS1 opsin downregulation. Smolts exhibited decreased accumulation of TRalpha and TRbeta transcripts in all quadrants, but this effect was most pronounced in the dorso-temporal (DT) retinal quadrant where UVS cones persist. By contrast, in 2 day T4-treated parr, the DT quadrant showed increased expression of TRalpha and TRbeta. Furthermore, D2 transcripts decreased in the DT quadrant of T4-treated parr but increased in the DT quadrant of smolts. These results suggest that T4 upregulates TRbeta expression to initiate SWS1 opsin downregulation, while TRalpha and TRbeta downregulation occurs to prevent natural loss of UVS cones from the DT retina.

Bax-Induced Apoptosis in Leber's Congenital Amaurosis: A Dual Role in Rod and Cone Degeneration

Pathogenesis in the Rpe65−/− mouse model of Leber's congenital amaurosis (LCA) is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65 −/− mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65), was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65−/− mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors.Quantitative PCR analysis showed that increased expression of pro-apoptotic Bax and decreased level of anti-apoptotic Bcl-2 were restored in Rpe65−/−/Gnat1−/− mice lacking the Gnat1 gene encoding rod transducin. Moreover, photoreceptor apoptosis was prevented as assessed by TUNEL assay. These data indicate that abnormal activity of opsin apoprotein induces retinal cell apoptosis through the Bcl-2-mediated pathway. Following immunohistological and real-time PCR analyses, we further observed that decreased expression of rod genes in Rpe65-deficient mice was rescued in Rpe65−/−/Bax−/− mice. Histological and TUNEL studies confirmed that rod cell demise and apoptosis in diseased Rpe65−/− mice were dependent on Bax-induced pathway. Surprisingly, early loss of cones was not prevented in Rpe65−/−/Bax−/− mice, indicating that pro-apoptotic Bax was not involved in the pathogenesis of cone cell death in Rpe65-deficient mice.This is the first report, to our knowledge, that a single genetic mutation can trigger two independent apoptotic pathways in rod and cone photoreceptors in Rpe65-dependent LCA disease. These results highlight the necessity to investigate and understand the specific death signaling pathways committed in rods and cones to develop effective therapeutic approaches to treat RP diseases.

Mapping Retinal Degeneration and Loss-of-Function inRd-FTLMice

Retinitis pigmentosa (RP) is a blinding disease caused by the degeneration of photoreceptors. To further understand the process of degeneration in RP, the authors have traced activation patterns associated with rod and cone photoreceptor degeneration in a mouse model of RP METHODS: The authors used a double-mutant mouse, Rd-FTL, which contains a natural mutation, rd1, affecting the rod photoreceptors and an axon-targeted beta-galactosidase reporter system, which is under the regulation of the promoter of the c-fos gene. These mice allowed the authors to trace degeneration-related activity that corresponded to rod and cone death. The authors traced cell death-associated activation in both rods and cones, allowing them to accurately determine the time course of cone degeneration in these mice. In the analysis of downstream activation patterns in the inner retina, they found that amacrine and ganglion cells maintain their photopic light-related activation until at least the initiation of cone degeneration. These cell populations then show increased activity during the peak time of cone cell degeneration. The authors also examined light-regulated functional activation of a subclass of amacrine cells, the dopaminergic amacrine cells. These cells showed light-induced functional activation after rod photoreceptor death and until the period of cone photoreceptor death, suggesting that they can be regulated by cone photoreceptors alone. These findings have helped to accurately trace the periods of photoreceptor degeneration in this model of RP and show that correct light-regulated inner retinal activation is maintained until the time of cone degeneration.

RPGR ORF15 genotype and clinical variability of retinal degeneration in an Australian population

Background:Mutations in the retinitis pigmentosa GTPase regulator gene (RPGR) are estimated to cause up to 20% of all Caucasian retinitis pigmentosa and up to 75% of cases of X-Linked RP...

Functional Cone Rescue by RdCVF Protein in a Dominant Model of Retinitis Pigmentosa

In retinitis pigmentosa (RP), a majority of causative mutations affect genes solely expressed in rods; however, cone degeneration inevitably follows rod cell loss. Following transplantation and in vitro studies, we demonstrated the role of photoreceptor cell paracrine interactions and identified a Rod-derived Cone Viability Factor (RdCVF), which increases cone survival. In order to establish the clinical relevance of such mechanism, we assessed the functional benefit afforded by the injection of this factor in a frequent type of rhodopsin mutation, the P23H rat. In this model of autosomal dominant RP, RdCVF expression decreases in parallel with primary rod degeneration, which is followed by cone loss. RdCVF protein injections induced an increase in cone cell number and, more important, a further increase in the corresponding electroretinogram (ERG). These results indicate that RdCVF can not only rescue cones but also preserve significantly their function. Interestingly, the higher amplitude of the functional versus the survival effect of RdCVF on cones indicates that RdCVF is acting more directly on cone function. The demonstration at the functional level of the therapeutic potential of RdCVF in the most frequent of dominant RP mutations paves the way toward the use of RdCVF for preserving central vision in many RP patients.

Functional and structural assessment of retinal sheet allograft transplantation in feline hereditary retinal degeneration

To investigate whether sheets of fetal retinal allografts can integrate into the dystrophic Abyssinian cat retina with progressive rod cone degeneration. Fetal retinal sheets (cat gestational day 42), incubated with BDNF microspheres, were transplanted to the subretinal space of four cats at an early disease stage. Cats were studied by fundus examinations, bilateral full-field flash ERGs, and indocyanine green and fluorescein angiograms up to 4 months following surgery. E42 donor and transplanted eyes were analyzed by histology and immunohistochemistry for retinal markers. Funduscopy and angiography showed good integration of the transplants in two of four cats, including extension of host blood vessels into the transplant and some scarring in the host. In these two, transplants were found in the subretinal space with laminated areas, with photoreceptor outer segments in normal contacts with the host retinal pigment epithelium. In some areas, transplants appeared to be well-integrated within the host neural retina. Neither of these two cats showed functional improvement in ERGs. In the other two cats, only remnants of donor tissue were left. Transplants stained for all investigated cellular markers. No PKC immunoreactivity was detected in the fetal donor retina at E42, but developed in the 4-month-old grafts. Fetal sheet transplants can integrate well within a degenerating cat retina and develop good lamination of photoreceptors. Functional improvement was not demonstrated by ERG in cats with well-laminated grafts. Transplants need to be further evaluated in cat host retinas with a more advanced retinal degeneration using longer follow-up times.

ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

To characterize the ZBED4 cDNA identified by subtractive hybridization and microarray of retinal cone degeneration (cd) adult dog mRNA from mRNA of normal dog retina. The cDNA library obtained from subtractive hybridization was arrayed and screened with labeled amplicons from normal and cd dog retinas. Northern blot analysis was used to verify ZBED4 mRNA expression in human retina. Flow cytometry sorted peanut agglutinin (PNA)-labeled cones from dissociated mouse retinas, and quantitative RT-PCR (QPCR) was used to measure ZBED4 mRNA levels in these cone cells. Immunohistochemistry localized ZBED4 in human retinas. Expression of ZBED4 mRNA transiently transfected into HEK293 cells was analyzed by immunofluorescence. ZBED4 subcellular localization was determined with Western blot analysis. One of 80 cDNAs differentially expressed in normal and cd dog retinas corresponded to a novel gene, ZBED4, which is also expressed in human and mouse retinas. ZBED4 mRNA was found to be present in cone photoreceptors. When ZBED4 cDNA was transfected into HEK293 cells, the expressed protein showed nuclear localization. However, in human retinas, ZBED4 was localized to cone nuclei, inner segments, and pedicles, as well as to Müller cell endfeet. Confirming these immunohistochemical results, the 135-kDa ZBED4 was found in both the nuclear and cytosolic extracts of human retinas. ZBED4 has four predicted DNA-binding domains, a dimerization domain, and two LXXLL motifs characteristic of coactivators/corepressors of nuclear hormone receptors. ZBED4 cellular/subcellular localization and domains suggest a regulatory role for this protein, which may exert its effects in cones and Müller cells through multiple ways of action.

Cone Degeneration in Aging and Age-Related Macular Degeneration

To examine the morphological features of macular photoreceptors in histologically normal retina from normal donor eyes and eyes with age-related macular degeneration (AMD). The macular region was excised from 18 donor eyes (aged 22-96 years) and cryosectioned. Sections were stained with hematoxylin-eosin or double immunolabeled using opsin antibodies or synaptic markers. Three of 8 retinas studied in detail had AMD lesions; the remainder were histologically normal. Immunoreactivity to cone opsin was abnormal in parts of all retinas (3.5%-95.0% of each sample) and was associated with swelling of and altered immunoreactivity in the cone distal axon. In non-AMD retinas, the anomalies were mainly in nonfoveal macular locations. The nature of the anomalies was identical in non-AMD retinas and in parts of AMD retinas adjacent to overt degeneration. Redistribution of opsin and anomalies in the distal cone axon are common in the aging human macula and may indicate susceptibility to AMD. The findings are consistent with tests of cone function in aging and early AMD, which suggests that integrated cone functions--including contrast sensitivity, color matching, and short wavelength-sensitive cone sensitivity--are the most reliable prognostic indicators of progression in AMD.

Loss of cone photoreceptors caused by chromophore depletion is partially prevented by the artificial chromophore pro-drug, 9-cis-retinyl acetate

Inactivating mutations in the retinoid isomerase (RPE65) or lecithin:retinol acyltransferase (LRAT) genes cause Leber congenital amaurosis (LCA), a severe visual impairment in humans. Both enzymes participate in the retinoid (visual) cycle, the enzymatic pathway that continuously generates 11-cis-retinal, the chromophore of visual pigments in rod and cone photoreceptor cells needed for vision. We investigated human RPE65-LCA patients and mice with visual cycle abnormalities to determine the impact of chronic chromophore deprivation on cones. Young patients with RPE65 mutations showed foveal cone loss along with shortened inner and outer segments of remaining cones; cone cell loss also was dramatic in young mice lacking Rpe65 or Lrat gene function. To selectively evaluate cone pathophysiology, we eliminated the rod contribution to electroretinographic (ERG) responses by generating double knockout mice lacking Lrat or Rpe65 together with an inactivated rod-specific G protein transducin gene (Gnat1-/-). Cone ERG responses were absent in Gnat1-/-Lrat-/- mice which also showed progressive degeneration of cones. Cone ERG responses in Gnat1-/-Rpe65-/- mice were markedly reduced and declined over weeks. Treatment of these mice with the artificial chromophore pro-drug, 9-cis-retinyl acetate, partially protected inferior retinal cones as evidenced by improved ERGs and retinal histochemistry. Gnat1-/- mice chronically treated with retinylamine, a selective inhibitor of RPE65, also showed a decline in the number of cones that was ameliorated by 9-cis-retinyl acetate. These results suggest that chronic lack of chromophore leads to progressive loss of cones in mice and humans. Therapy for LCA patients should be geared toward early adequate delivery of chromophore to cone photoreceptors.

Early remodeling in an inducible animal model of retinal degeneration

Photoreceptor degeneration is followed by significant morphological changes in the second-order retinal neurons in humans and in several genetic animal models. However, it is not clear whether similar changes occur when photoreceptor degeneration is induced nongenetically, raising the question whether these changes are a general effect of deafferentation independent of the cause of degeneration. We addressed this by inducing selective photoreceptor degeneration with N-methyl- N-nitrosourea (MNU) and studying its effects on inner retinal neurons in a mouse for up to 3 months, using immunocytochemistry and iontophoretic labeling. To develop objective measures of photoreceptor degeneration and of retinal remodeling, we measured several retinal proteins using immunoblot analysis, and quantified gross visual ability of the animal in a visual cliff test. The MNU-induced progressive degeneration of rods and cones was associated with declining levels of postsynaptic density 95 protein in the retina, and with deteriorating visual performance of the animal. Müller glial cells showed enhanced reactivity for glial fibrillary acidic protein as demonstrated by immunocytochemistry, which also reflected in increased levels of the protein as demonstrated by immunoblotting. Horizontal cells and rod bipolar cells progressively lost their dendritic processes, which correlated with a slight decline in the levels of calbindin and protein kinase C alpha respectively. Horizontal cell axons, immunoreactive for nonphosphorylated neurofilaments, showed sprouting into the inner nuclear layer. Ganglion cells and their synaptic inputs, probed by immunolocalizing β-III- tubulin, neurofilaments, bassoon and synaptophysin, appeared to be unaffected. These results demonstrate that MNU-induced photoreceptor degeneration leads to retinal remodeling similar to that observed in genetic models, suggesting that the remodeling does not depend on the etiopathology that underlies photoreceptor degeneration.

Retinal Ganglion Cells Survive and Maintain Normal Dendritic Morphology in a Mouse Model of Inherited Photoreceptor Degeneration

Retinitis pigmentosa (RP), a family of inherited disorders characterized by progressive photoreceptor death, is a leading cause of blindness with no available cure. Despite the genetic heterogeneity underlying the disease, recent data on animal models show that the degeneration of photoreceptors triggers stereotyped remodeling among their postsynaptic partners. In particular, bipolar and horizontal cells might undergo dendritic atrophy and secondary death. The aim of this study was to investigate whether or not concomitant changes also occur in retinal ganglion cells (RGCs), the only retinal projection neurons to the brain and the proposed substrate for various therapeutic approaches for RP. We assessed the retention of morphology, overall architecture, and survival of RGCs in a mouse model of RP at various stages of the disease. To study the morphology of single RGCs, we generated a new mouse line by crossing Thy1-GFP-M mice (Feng et al., 2000), which express GFP (green fluorescent protein) in a small number of heterogeneous RGCs types, and rd10 mutants, a model of autosomal recessive RP, which exhibit a typical rod-cone degeneration (Chang et al., 2002). We show remarkable preservation of RGC structure, survival, and projections to higher visual centers in the time span from 3 to 9 months of life, well beyond the death of photoreceptors. Thus, unlike second-order neurons, RGCs appear as a considerably stable population of cells, potentially constituting a favorable substrate for restoring vision in RP individuals by means of electronic prostheses or direct expression of photosensitive proteins.