Cone Cell Loss Research Articles

LONGITUDINAL ADAPTIVE OPTICS SCANNING LASER OPHTHALMOSCOPY REVEALS REGIONAL VARIATION IN CONE AND ROD PHOTORECEPTOR LOSS IN STARGARDT DISEASE.

To investigate the temporal sequence of changes in the photoreceptor cell mosaic in patients with Stargardt disease type 1, using adaptive optics scanning laser ophthalmoscopy. Two brothers with genetically confirmed Stargardt disease type 1 underwent comprehensive eye exams, spectral-domain optical coherence tomography, fundus autofluorescence, and adaptive optics scanning laser ophthalmoscopy imaging 3 times over the course of 28 months. Confocal images of the cones and rods were obtained from the central fovea to 10° inferiorly. Photoreceptors were counted in sampling windows at 100- µ m intervals of 200 µ m × 200 µ m for cones and 50 µ m × 50 µ m for rods, using custom cell marking software with manual correction. Photoreceptor density and spacing were measured and compared across imaging sessions using one-way analysis of variance. Adaptive optics scanning laser ophthalmoscopy revealed the younger brother had a 30% decline in foveal cone density after 8 months, followed by complete loss of foveal cones at 28 months; the older brother had no detectable foveal cones at baseline. In the peripheral macula, cone and rod spacings were greater than normal in both patients. The ratio of the cone spacing to rod spacing was greater than normal across all eccentricities, with a greater divergence closer to the foveal center. Cone cell loss may be an early pathogenetic step in Stargardt disease. Adaptive optics scanning laser ophthalmoscopy provides the capability to track individual photoreceptor changes longitudinally in Stargardt disease.

Short-term Assessment of Subfoveal Injection of Adeno-Associated Virus-Mediated hCHM Gene Augmentation in Choroideremia Using Adaptive Optics Ophthalmoscopy

Subretinal injection for gene augmentation in retinal degenerations forcefully detaches the neural retina from the retinal pigment epithelium, potentially damaging photoreceptors and/or retinal pigment epithelium cells. To use adaptive optics scanning light ophthalmoscopy (AOSLO) to assess the short-term integrity of the cone mosaic following subretinal injections of adeno-associated virus vector designed to deliver a functional version of the CHM gene (AAV2-hCHM) in patients with choroideremia. This longitudinal case series study enrolled adult patients with choroideremia from February 2015 to January 2016 in the US. To be included in the study, study participants must have received uniocular subfoveal injections of low-dose (5 × 1010 vector genome per eye) or high-dose (1 × 1011 vector genome per eye) AAV2-hCHM. Analysis began February 2015. The macular regions of both eyes were imaged before and 1 month after injection using a custom-built multimodal AOSLO. Postinjection cone inner segment mosaics were compared with preinjection mosaics at multiple regions of interest. Colocalized spectral-domain optical coherence tomography and dark-adapted cone sensitivity was also acquired at each time point. Nine study participants ranged in age from 26 to 50 years at the time of enrollment, and all were White men. Postinjection AOSLO images showed preservation of the cone mosaic in all 9 AAV2-hCHM-injected eyes. Mosaics appeared intact and contiguous 1 month postinjection, with the exception of foveal disruption in 1 patient. Optical coherence tomography showed foveal cone outer segment shortening postinjection. Cone-mediated sensitivities were unchanged in 8 of 9 injected and 9 of 9 uninjected eyes. One participant showed acute loss of foveal optical coherence tomography cone outer segment-related signals along with cone sensitivity loss that colocalized with disruption of the mosaic on AOSLO. Integrity of the cone mosaic is maintained following subretinal delivery of AAV2-hCHM, providing strong evidence in support of the safety of the injections. Minor foveal thinning observed following surgery corresponds with short-term cone outer segment shortening rather than cone cell loss. Foveal cone loss in 1 participant raises the possibility of individual vulnerability to the subretinal injection.

Analysis of Early Cone Dysfunction in an In Vivo Model of Rod-Cone Dystrophy.

Retinitis pigmentosa (RP) is a generic term for a group of genetic diseases characterized by loss of rod and cone photoreceptor cells. Although the genetic causes of RP frequently only affect the rod photoreceptor cells, cone photoreceptors become stressed in the absence of rods and undergo a secondary degeneration. Changes in the gene expression profile of cone photoreceptor cells are likely to occur prior to observable physiological changes. To this end, we sought to achieve greater understanding of the changes in cone photoreceptor cells early in the degeneration process of the Rho−/− mouse model. To account for gene expression changes attributed to loss of cone photoreceptor cells, we normalized PCR in the remaining number of cones to a cone cell reporter (OPN1-GFP). Gene expression profiles of key components involved in the cone phototransduction cascade were correlated with tests of retinal cone function prior to cell loss. A significant downregulation of the photoreceptor transcription factor Crx was observed, which preceded a significant downregulation in cone opsin transcripts that coincided with declining cone function. Our data add to the growing understanding of molecular changes that occur prior to cone dysfunction in a model of rod-cone dystrophy. It is of interest that gene supplementation of CRX by adeno-associated viral vector delivery prior to cone cell loss did not prevent cone photoreceptor degeneration in this mouse model.

Vascular dysfunction in retinitis pigmentosa.

The relationship between ocular haemodynamics and retinitis pigmentosa (RP) has not been fully understood. Reductions in blood flow have been established in RP patients by a variety of studies; however, questions have yet to be answered regarding the role of vascular dysfunction in photoreceptors (PR) degeneration, the causes of vascular dysfunction in RP, as well as the diagnostic, prognostic and perhaps therapeutic potential of measuring ocular haemodynamics in RP patients. While significant evidence supports the theory that vascular dysfunction is associated with but not the cause of PR death in retinitis pigmentosa, evidence suggests that vascular abnormalities in the foveal and parafoveal regions may exacerbate cone cell loss. Additional evidence demonstrates that vascular dysfunction likely results from changes in metabolic demand due to death of PR cells in the retina. Detection and monitoring of ocular blood flow, retinal oxygen saturation, endothelin-1 levels and vascular structural abnormalities could provide diagnostic, prognostic and therapeutic potential for patients with RP.

Targeted next generation sequencing identified novel loss-of-function mutations in MERTK gene in Chinese patients with retinitis pigmentosa.

BackgroundRetinitis pigmentosa (RP) is one of the major types of hereditary retinal dystrophies with extreme genotypic heterogeneity. To date, more than 80 genes have been identified to be associated with RP in human.MethodHere, we presented a clinical genetic study of three Chinese man manifested with night vision blindness and complete loss of midperipheral visual field. All of these three probands have been identified with loss of both central vision and far peripheral visual field. Gradual loss of rod cells followed by subsequent loss of cone cells have been identified in these probands. Targeted next generation sequencing and Sanger sequencing have been performed to understand the pathogenic variants underlying the disease phenotype in these three unrelated Chinese probands.ResultsTargeted next generation sequencing and Sanger sequencing identified three homozygous novel mutations (c.1880C>A; c.1459_1460delGA, and c.392G>A) in the MERTK gene in these three unrelated Chinese proband. In the first proband, the identified mutation (c.1880C>A) leads to the formation of a premature stop codon followed by the formation of a truncated mer‐tyrosine kinase (MERTK) protein (p.Ser627*) product which predicted to be disease causing. In the second proband, the identified deletion (c.1459_1460delGA) leads to the formation of a frameshift which also finally results in the formation of a truncated MERTK protein (p.Asp487Leufs*57) product which also predicted to be disease causing. In the third proband, the identified mutation (c.392G>A) leads to the formation of a premature stop codon followed by the formation of a truncated MERTK protein (p.Trp131*) product which also predicted to be disease causing. Hence, these three mutations are loss‐of‐function mutations. These three mutations were absent in unaffected family members and in 100 normal healthy controls.ConclusionOur present study also demonstrates the significance of targeted next generation sequencing in determining the genetic basis of RP.

Regulation of Notch signaling in the developing Drosophila eye by a T-box containing transcription factor, Dorsocross.

Owing to a multitude of functions, there is barely a tissue or a cellular process that is not being regulated by Notch signaling. To allow the Notch signal to be deployed in numerous contexts, many different mechanisms have evolved to regulate the level, duration and spatial distribution of Notch activity. To identify novel effectors of Notch signaling in Drosophila melanogaster, we analyzed the whole transcriptome of the wing and eye imaginal discs in which an activated form of Notch was overexpressed. Selected candidate genes from the transcriptome analysis were subjected to genetic interaction experiments with Notch pathway components. Among the candidate genes, T-box encoding gene, Dorsocross (Doc) showed strong genetic interaction with Notch ligand, Delta. Genetic interaction between them resulted in reduction of eye size, loss of cone cells, and cell death, which represent prominent Notch loss of function phenotypes. Immunocytochemical analysis in Df(3L)DocA/Dl 5f trans-heterozygous eye discs showed accumulation of Notch at the membrane. This accumulation led to decreased Notch signaling activity as we found downregulation of Atonal, a Notch target and reduction in the rate of Notch-mediated cell proliferation. Doc mutant clones generated by FLP-FRT system showed depletion in the expression of Delta and subsequent reduction in the Notch signaling activity. Similarly, Doc overexpression in the eye discs led to modification of Delta expression, loss of Atonal expression and absence of eye structure in pharate adults. Taken together, our results suggest that Doc regulates the expression of Delta and influence the outcome of Notch signaling in the eye discs.

Cell Death Pathways in Mutant Rhodopsin Rat Models Identifies Genotype-Specific Targets Controlling Retinal Degeneration.

Retinitis pigmentosa (RP) is a group of inherited neurological disorders characterized by rod photoreceptor cell death, followed by secondary cone cell death leading to progressive blindness. Currently, there are no viable treatment options for RP. Due to incomplete knowledge of the molecular signaling pathways associated with RP pathogenesis, designing therapeutic strategies remains a challenge. In particular, preventing secondary cone photoreceptor cell loss is a key goal in designing potential therapies. In this study, we identified the main drivers of rod cell death and secondary cone loss in the transgenic S334ter rhodopsin rat model, tested the efficacy of specific cell death inhibitors on retinal function, and compared the effect of combining drugs to target multiple pathways in the S334ter and P23H rhodopsin rat models. The primary driver of early rod cell death in the S334ter model was a caspase-dependent process, whereas cone cell death occurred though RIP3-dependent necroptosis. In comparison, rod cell death in the P23H model was via necroptotic signaling, whereas cone cell loss occurred through inflammasome activation. Combination therapy of four drugs worked better than the individual drugs in the P23H model but not in the S334ter model. These differences imply that treatment modalities need to be tailored for each genotype. Taken together, our data demonstrate that rationally designed genotype-specific drug combinations will be an important requisite to effectively target primary rod cell loss and more importantly secondary cone survival.

The mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye.

Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling mediated by Epidermal growth factor receptor (EGFR) and the Sevenless (Sev) receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of miR-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. miR-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently derepresses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev, which otherwise completely lack this cell fate. Altogether, we reveal a rare setting of developmental cell specification that involves substantial miRNA control.

Loss of Tmem30a leads to photoreceptor degeneration

Phosphatidylserine (PS) is asymmetrically distributed between the outer and inner leaflets of the plasma membrane in eukaryotic cells. PS asymmetry on the plasma membrane depends on the activities of P4-ATPases, and disruption of PS distribution can lead to various disease conditions. Folding and transporting of P4-ATPases to their cellular destination requires the β subunit TMEM30A proteins. However, the in vivo functions of Tmem30a remain unknown. To this end, we generated retinal-specific Tmem30a-knockout mice to investigate its roles in vivo for the first time. Our data demonstrated that loss of Tmem30a in mouse cone cells leads to mislocalization of cone opsin, loss of photopic electroretinogram (ERG) responses and loss of cone cells. Mechanistically, Tmem30a-mutant mouse embryonic fibroblasts (MEFs) exhibited diminished PS flippase activity and increased exposure of PS on the cell surface. The broad loss of Tmem30a in adult mice led to a reduced scotopic photoresponse, mislocalization of ATP8A2 to the inner segment and cell body, and increased apoptosis in the retina. Our data demonstrated novel essential roles of Tmem30a in the retina.

Quantitative and Topographical Analysis of the Losses of Cone Photoreceptors and Retinal Ganglion Cells Under Taurine Depletion.

Taurine depletion is known to induce photoreceptor degeneration and was recently found to also trigger retinal ganglion cell (RGC) loss similar to the retinal toxicity of vigabatrin. Our objective was to study the topographical loss of RGCs and cone photoreceptors, with a distinction between the two cone types (S- and L- cones) in an animal model of induced taurine depletion. We used the taurine transporter (Tau-T) inhibitor, guanidoethane sulfonate (GES), to induce taurine depletion at a concentration of 1% in the drinking water. Spectral-domain optical coherence tomography (SD-OCT) and electroretinograms (ERG) were performed on animals after 2 months of GES treatment administered through the drinking water. Retinas were dissected as wholemounts and immunodetection of Brn3a (RGC), S-opsin (S-cones), and L-opsin (L-cones) was performed. The number of Brn3a+ RGCs, and L- and S-opsin+ cones was automatically quantified and their retinal distribution studied using isodensity maps. The treatment resulted in a significant reduction in plasma taurine levels and a profound dysfunction of visual performance as shown by ERG recordings. Optical coherence tomography analysis revealed that the retina was thinner in the taurine-depleted group. S-opsin+cones were more affected (36%) than L-opsin+cones (27%) with greater cone cell loss in the dorsal area whereas RGC loss (12%) was uniformly distributed. This study confirms that taurine depletion causes RGC and cone loss. Electroretinograms results show that taurine depletion induces retinal dysfunction in photoreceptors and in the inner retina. It establishes a gradient of cell loss depending on the cell type from S-opsin+cones, L-opsin+cones, to RGCs. The greater cell loss in the dorsal retina and of the S-cone population may underline different cellular mechanisms of cellular degeneration and suggests that S-cones may be more sensitive to light-induced retinal toxicity enhanced by the taurine depletion.

270. Optogenetic Activation of Remnant Cone Cells Can Restore Ganglion Cell Light Responses in a Canine Model of Retinitis Pigmentosa

In retinitis pigmentosa, the rod photoreceptor cells are generally lost first followed by a slow loss of cone cells. Remnant cone cell bodies have been observed even after many years of vision loss in retinitis pigmentosa patients. Proof-of-principle studies in retinal degeneration mice have demonstrated vision restoration upon expression of the optogene halorhodopsin in remnant cone cells. However, whether this will work in a large animal model of retinal degeneration is unclear. Here we look at light responses and histology post-treatment with halorhodopsin in a dog model of autosomal recessive retinitis pigmentosa. Halorhodopsin was expressed in the cone cells unilaterally in rcd1 dogs (PDE6β mutation) with mid- to late stage retinal degeneration using recombinant adeno-associated viral vectors. The retinas were imaged using OCT and function was evaluated using ERG and pupillometry. The spiking activity of retinal ganglion cells (RGCs) in retinal patches adjacent to and distant from the point of injection was tested using multi electrode array recordings. Retinal patches were stimulated with 589 nm light of different durations/intensities. OCT revealed significant thinning of the photoreceptor layer in the retinas of the dogs. While the ERG and pupillometry data did not show a significant difference between the treated and untreated eyes, multi-electrode array recordings revealed a restoration of ganglion cell light responses (ON, OFF and ON/OFF-responses to discrete flashes and responses to 4Hz flicker, 589nm light) in the treated retinal patch compared to an untreated control patch of retina. The lowest intensities where responses become clearly detectable for two animals were 0.1 mW/cm2 and 2.8 mW/cm2 probably reflecting different levels of NpHR expression. We observed halorhodopsin expression by histology in the remnant cone cell bodies of the treated retinas. Restoration of ganglion cell responses in the treated retinal patches suggests that the retina can be sensitized to light even after major photoreceptor cell loss. Ongoing work will determine if this restoration of ganglion cell light responses in the dogs translate to being able to navigate in the light.

Deletion of the p85α Regulatory Subunit of Phosphoinositide 3-Kinase in Cone Photoreceptor Cells Results in Cone Photoreceptor Degeneration

Downregulation of the retinal insulin/mTOR pathway in mouse models of retinitis pigmentosa is linked to cone cell death, which can be delayed by systemic administration of insulin. A classic survival kinase linking extracellular trophic/growth factors with intracellular antiapoptotic pathways is phosphoinositide 3-kinase (PI3K), which the authors have shown to protect rod photoreceptors from stress-induced cell death. The role of PI3K in cones was studied by conditional deletion of its p85α regulatory subunit. Mice expressing Cre recombinase in cones were bred to mice with a floxed pi3k gene encoding the p85α regulatory subunit of the PI3K and were back-crossed to ultimately generate offspring with cone-specific p85α knockout (cKO). Cre expression and cone-specific localization were confirmed by Western blot analysis and immunohistochemistry (IHC), respectively. Cone structural integrity was determined by IHC using peanut agglutinin and an M-opsin-specific antibody. Electroretinography (ERG) was used to assess rod and cone photoreceptor function. Retinal structure was examined by light and electron microscopy. An age-related cone degeneration was found in cKO mice, evidenced by a reduction in photopic ERG amplitudes and loss of cone cells. By 12 months of age, approximately 78% of cones had died, and progressive disorganization of synaptic ultrastructure was noted in surviving cone terminals in cKO retinas. Rod viability was unaffected in p85α cKO mice. The present study suggests that PI3K signaling pathway is essential for cone survival in the mouse retina.

Progressive Loss of Cones in Achromatopsia: An Imaging Study Using Spectral-Domain Optical Coherence Tomography

Achromatopsia (ACHM) is a congenital autosomal recessive cone disorder with a presumed stationary nature and only a few causative genes. Animal studies suggest that ACHM may be a good candidate for corrective gene therapy. Future implementation of this therapy in humans requires the presence of viable cone cells in the retina. In this study the presence of cone cells in ACHM was determined, as a function of age. The appearance and thickness of all retinal layers were evaluated by spectral-domain optical coherence tomography (SD-OCT) in 40 ACHM patients (age range, 4-70 years) with known mutations in the CNGB3, CNGA3, and PDE6C genes. A comparison was made with 55 healthy age-matched control subjects. The initial feature of cone cell decay was loss of inner and outer segments with disruption of the ciliary layer on OCT, which was observed as early as 8 years of age. Cone cell loss further progressed with age and occurred in 8 (42%) of 19 patients below 30 years and in 20 (95%) of 21 of those aged 30+ years. Retinal thickness was significantly thinner in the fovea of all patients (126 μm in ACHM vs. 225 μm in the control; P < 0.001) and correlated with age (β = 0.065; P = 0.011). Foveal hypoplasia was present in 24 (80%) of 30 patients and in 1 of 55 control subjects. ACHM is not a stationary disease. The first signs of cone cell loss occur in early childhood. If intervention becomes available in the future, the present results imply that it should be applied in the first decade.

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.

Activation of Bone Marrow-Derived Microglia Promotes Photoreceptor Survival in Inherited Retinal Degeneration

The role of microglia in neurodegeneration is controversial, although microglial activation in the retina has been shown to provide an early response against infection, injury, ischemia, and degeneration. Here we show that endogenous bone marrow (BM)-derived microglia play a protective role in vascular and neural degeneration in the retinitis pigmentosa model of inherited retinal degeneration. BM-derived cells were recruited to the degenerating retina where they differentiated into microglia and subsequently localized to the degenerating vessels and neurons. Inhibition of stromal-derived factor-1 in the retina reduced the number of BM-derived microglia and accelerated the rate of neurovascular degeneration. Systemic depletion of myeloid progenitors also accelerated the degenerative process. Conversely, activation of BM-derived myeloid progenitors by systemic administration of both granulocyte colony-stimulating factor and erythropoietin resulted in the deceleration of retinal degeneration and the promotion of cone cell survival. These data indicate that BM-derived microglia may play a protective role in retinitis pigmentosa. Functional activation of BM-derived myeloid progenitors by cytokine therapy may provide a novel strategy for the treatment of inherited retinal degeneration and other neurodegenerative diseases, regardless of the underlying genetic defect.

Deubiquitylating enzyme UBP64 controls cell fate through stabilization of the transcriptional repressor tramtrack.

Protein ubiquitylation plays a central role in multiple signal transduction pathways. However, the substrate specificity and potential developmental roles of deubiquitylating enzymes remain poorly understood. Here, we show that the Drosophila ubiquitin protease UBP64 controls cell fate in the developing eye. UBP64 represses neuronal cell fate but promotes the formation of nonneuronal cone cells. Using a proteomics approach, we identified the transcriptional repressor Tramtrack (TTK) as a primary UBP64 substrate. In common with TTK, reduced UBP64 levels lead to a loss of cone cells, supernumerary photoreceptors, and mechanosensory bristle cells. Previously, it was demonstrated that the blockade of neuronal cell fate was relieved by SINA-dependent ubiquitylation and degradation of TTK. We found that UBP64 counteracts SINA function by deubiquitylating TTK, leading to its stabilization and thereby promoting a nonneuronal cell fate. Mass spectrometric mapping revealed that SINA ubiquitylates multiple sites dispersed throughout TTK, which are duly deubiquitylated by UBP64. This observation suggests that both E3 SINA and UBP64 use a scanning mechanism to (de)ubiquitylate TTK. We conclude that the balance of TTK ubiquitylation by SINA and deubiquitylation by UBP64 constitutes a specific posttranslational switch controlling cell fate.

Delayed Loss of Cone and Remaining Rod Photoreceptor Cells due to Impairment of Choroidal Circulation after Acute Light Exposure in Rats

To examine the long-term effects of acute photooxidative stress in the retina, retinal pigment epithelium (RPE), and choroid. Albino rats injected with either the protective antioxidant phenyl-N-tert-butylnitrone (PBN) or saline 30 minutes before exposure to 5 klx white fluorescent light for 6 hours were kept for up to 3 months in 5 lux cyclic light. Electroretinograms were recorded, and the outer nuclear layer (ONL) and the choroidal thickness and area were measured after hematoxylin-eosin (H&E) staining. The expression of rod, cone, and RPE cell markers was detected by Western blotting, and apoptosis was analyzed by TUNEL staining. Oxidative stress was analyzed by immunohistochemistry against 4-hydroxynonenal (4-HNE)-modified proteins. Retinal and choroidal ultrastructures were observed by transmission electron microscopy (TEM). Choroidal circulation was analyzed by in vivo staining of the choroidal layer by trypan blue. In the saline-injected animals, TUNEL- and 4-HNE-labeling in the ONL, RPE, and choroid were higher 24 hours and 7 days after light exposure, and ERG amplitude, ONL and choroidal thickness and area, and rhodopsin and RPE65 expression were lower 7 or more days after light exposure than in phenyl-N-tert-butylnitrone (PBN)-injected animals. In the saline-injected animals, the expression of mid-wavelength opsin and the presence of cone cells in the ONL and the choroidal circulation were preserved for 7 days after light exposure but started to decrease by 1 month and continued to decrease for 3 months after light exposure. An increase in TUNEL-positive cells was observed in the ONL at the inferior peripheral retina, just behind the iris, by 3 months after light exposure. Delayed loss of cone cells, remaining rod cells, and choroidal circulation were counteracted by PBN treatment. Although cone cells are resistant to cell damage induced by acute photooxidative stress, progressive loss of cone cells continued for up to 3 months after light exposure. Impaired choroidal circulation is likely to be involved in the mechanism of delayed photoreceptor cell death after light exposure. Preserving choroidal circulation may provide a novel target for preserving the cone and the remaining rod cells in patients with retinal degeneration such as retinitis pigmentosa.

Mutations in rugose promote cell type-specific apoptosis in the Drosophila eye

RUGOSE (RG): encodes an A kinase anchor protein and was isolated as a genetic interactor of the Notch and epidermal growth factor receptor (EGFR) pathways during eye development in Drosophila. rg mutants display a small, rough eye phenotype primarily caused by the loss of cone cells. Here we show that the basis of this phenotype is cell type-specific apoptosis rather than transformation and hence can be rescued by reduction of proapoptotic signals. Moreover, a nearly complete rescue is observed by an increased Notch signal suggesting an antiapoptotic function of Notch in this developmental context. Cone cell loss in rg mutants is accompanied by enhanced Jun N-terminal kinase activity and, concomitantly, by a reduction of EGFR signalling activity. Together, these findings support the idea that rg plays an important role in the integration of different signals required for the exact regulation of cone cell development and survival.

Cone cell survival and downregulation of GCAP1 protein in the retinas of GC1 knockout mice.

To examine the spatial and temporal characteristics of cone cell survival and the expression of guanylate cyclase-activating proteins (GCAPs) in the guanylate cyclase (GC)-1 knockout (KO) mouse retina. Immunohistochemical analyses with peanut agglutinin and an antibody specific for cone transducin were used to examine cone cell survival in the GC1 KO retina at 4, 5, 9, 16, and 24 weeks of age. Immunohistochemical and Northern and Western blot analyses were used to examine the expression of GCAP1 and GCAP2 in 4- to 5-week-old mice. The number of cone cells appeared normal throughout the superior and inferior retinal regions in 4- and 5-week-old GC1 KO mice but gradually decreased by 6 months. Cone cell loss was exacerbated in the inferior retinal region, with only 2% to 8% remaining by 6 months of age; however, 40% to 70% of the cone cells survived in the superior region at this age. GCAP1 and GCAP2 protein levels were downregulated in GC1 KO retinas at 4 weeks of age and GCAP1 immunostaining was absent from the photoreceptor outer segments. The results of this study show that the rate of cone cell loss in the GC1 KO mouse is comparable to that previously described in the GUCY1*B chicken and in humans with Leber congenital amaurosis (LCA)-1. The GCAP expression data, when combined with those of previous electrophysiological studies of the GC1 KO mouse retina, provide evidence that GC1-GCAP1 interactions are essential for cone cell function in mice and that GC2 and GCAP2 activities contribute to the rod cell response in the absence of GC1.

Rugose (rg), a Drosophila A kinase anchor protein, is required for retinal pattern formation and interacts genetically with multiple signaling pathways.

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways.