Cone Degeneration Research Articles

Astrocytes and Müller Cell Alterations During Retinal Degeneration in a Transgenic Rat Model of Retinitis Pigmentosa.

Purpose: Retinitis pigmentosa includes a group of progressive retinal degenerative diseases that affect the structure and function of photoreceptors. Secondarily to the loss of photoreceptors, there is a reduction in retinal vascularization, which seems to influence the cellular degenerative process. Retinal macroglial cells, astrocytes, and Müller cells provide support for retinal neurons and are fundamental for maintaining normal retinal function. The aim of this study was to investigate the evolution of macroglial changes during retinal degeneration in P23H rats.Methods: Homozygous P23H line-3 rats aged from P18 to 18 months were used to study the evolution of the disease, and SD rats were used as controls. Immunolabeling with antibodies against GFAP, vimentin, and transducin were used to visualize macroglial cells and cone photoreceptors.Results: In P23H rats, increased GFAP labeling in Müller cells was observed as an early indicator of retinal gliosis. At 4 and 12 months of age, the apical processes of Müller cells in P23H rats clustered in firework-like structures, which were associated with ring-like shaped areas of cone degeneration in the outer nuclear layer. These structures were not observed at 16 months of age. The number of astrocytes was higher in P23H rats than in the SD matched controls at 4 and 12 months of age, supporting the idea of astrocyte proliferation. As the disease progressed, astrocytes exhibited a deteriorated morphology and marked hypertrophy. The increase in the complexity of the astrocytic processes correlated with greater connexin 43 expression and higher density of connexin 43 immunoreactive puncta within the ganglion cell layer (GCL) of P23H vs. SD rat retinas.Conclusions: In the P23H rat model of retinitis pigmentosa, the loss of photoreceptors triggers major changes in the number and morphology of glial cells affecting the inner retina.

Necrotic enlargement of cone photoreceptor cells and the release of high-mobility group box-1 in retinitis pigmentosa.

Retinitis pigmentosa (RP) refers to a group of inherited retinal degenerations resulting form rod and cone photoreceptor cell death. The rod cell death due to deleterious genetic mutations has been shown to occur mainly through apoptosis, whereas the mechanisms and features of the secondary cone cell death have not been fully elucidated. Our previous study showed that the cone cell death in rd10 mice, an animal model of RP, involves necrotic features and is partly mediated by the receptor interacting protein kinase. However, the relevancy of necrotic cone cell death in human RP patients remains unknown. In the present study, we showed that dying cone cells in rd10 mice exhibited cellular enlargement, along with necrotic changes such as cellular swelling and mitochondrial rupture. In human eyes, live imaging of cone cells by adaptive optics scanning laser ophthalmoscopy revealed significantly increased percentages of enlarged cone cells in the RP patients compared with the control subjects. The vitreous of the RP patients contained significantly higher levels of high-mobility group box-1, which is released extracellularly associated with necrotic cell death. These findings suggest that necrotic enlargement of cone cells is involved in the process of cone degeneration, and that necrosis may be a novel target to prevent or delay the loss of cone-mediated central vision in RP.

Long-term rescue of cone photoreceptor degeneration in retinitis pigmentosa 2 (RP2)-knockout mice by gene replacement therapy.

Retinal neurodegenerative diseases are especially attractive targets for gene replacement therapy, which appears to be clinically effective for several monogenic diseases. X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in RPGR or RP2 gene. With a goal to develop gene therapy for the XLRP-RP2 disease, we first performed detailed characterization of the Rp2-knockout (Rp2-KO) mice and observed early-onset cone dysfunction, which was followed by progressive cone degeneration, mimicking cone vision impairment in XLRP patients. The mice also exhibited distinct and significantly delayed falling phase of photopic b-wave of electroretinogram (ERG). Concurrently, we generated a self-complementary adeno-associated viral (AAV) vector carrying human RP2-coding sequence and demonstrated its ability to mediate stable RP2 protein expression in mouse photoreceptors. A long-term efficacy study was then conducted in Rp2-KO mice following AAV-RP2 vector administration. Preservation of cone function was achieved with a wide dose range over 18-month duration, as evidenced by photopic ERG and optomotor tests. The slower b-wave kinetics was also completely restored. Morphologically, the treatment preserved cone viability, corrected mis-trafficking of M-cone opsin and restored cone PDE6 expression. The therapeutic effect was achieved even in mice that received treatment at an advanced disease stage. The highest AAV-RP2 dose group demonstrated retinal toxicity, highlighting the importance of careful vector dosing in designing future human trials. The wide range of effective dose, a broad treatment window and long-lasting therapeutic effects should make the RP2 gene therapy attractive for clinical development.

Of ghrelin, cone cultivation, dinoflagellate eyes, and the cyanobacterial circadian clock.

Of ghrelin, cone cultivation, dinoflagellate eyes, and the cyanobacterial circadian clock.

CGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca(2+) channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3(-/-)/Nrl(-/-) mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca(2+) channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.

Gain and loss of function mutations of ataxin-7 cause cilia pathology in mouse and zebrafish models

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disease characterized by progressive neuronal loss in the cerebellum and associated structures, leading to cerebellar ataxia, dysarthria and dysphagia. Additional non-cerebellar symptoms are present at variable frequencies, including visual impairment affecting 83% of patients that is caused by cone and rod photoreceptor degeneration and hearing loss concerning 24% of patients. SCA7 is caused by a toxic polyglutamine expansion in Ataxin-7, a known subunit of the transcriptional co-regulator SAGA/TFTC complex. Transcriptional alterations are observed in SCA7, however, the disease mechanism is yet unclear. Ataxin-7 is also present in the cytoplasm of neurons, where its function is unknown. Here, we show for the first time that Ataxin-7 is a bona fide component of centrosomes and is present in diverse types of mammalian cilia, including primary, motile and photoreceptor sensory cilia. We provide evidence that Ataxin-7 loss of function causes a large spectrum of ciliary-related morphological and functional phenotypes in zebrafish, and that human Ataxin-7 is able to rescue the cilia phenotypes, demonstrating that Ataxin-7 has an essential, evolutionarily conserved function in cilia. Strikingly, Ataxin-7 is progressively lost from photoreceptor cilia in SCA7 mouse models, correlating with ciliary defects and photoreceptor degeneration. Finally, we found that expression of polyQ-expanded Ataxin-7, like Ataxin-7 loss of function, leads to ciliary dysfunctions in zebrafish. Our study thus provides new insight into the function of Ataxin-7 and opens novel perspective in the understanding of SCA7 pathogenesis.

Knockout ofRP2decreases GRK1 and rod transducin subunits and leads to photoreceptor degeneration in zebrafish

Retinitis pigmentosa (RP) affects about 1.8 million individuals worldwide. X-linked retinitis pigmentosa (XLRP) is one of the most severe forms of RP. Nearly 85% of XLRP cases are caused by mutations in the X-linked retinitis pigmentosa 2 (RP2) and RPGR. RP2 has been considered to be a GTPase activator protein for ARL3 and to play a role in the traffic of ciliary proteins. The mechanism of how RP2 mutations cause RP is still unclear. In this study, we generated an RP2 knockout zebrafish line using transcription activator-like effector nuclease technology. Progressive retinal degeneration could be observed in the mutant zebrafish. The degeneration of rods' outer segments (OSs) is predominant, followed by the degeneration of cones' OS. These phenotypes are similar to the characteristics of RP2 patients, and also partly consistent with the phenotypes of RP2 knockout mice and morpholino-mediated RP2 knockdown zebrafish. For the first time, we found RP2 deletion leads to decreased protein levels and abnormal retinal localizations of GRK1 and rod transducin subunits (GNAT1 and GNB1) in zebrafish. Furthermore, the distribution of the total farnesylated proteins in zebrafish retina is also affected by RP2 ablation. These molecular alterations observed in the RP2 knockout zebrafish might probably be responsible for the gradual loss of the photoreceptors' OSs. Our work identified the progression of retinal degeneration in RP2 knockout zebrafish, provided a foundation for revealing the pathogenesis of RP caused by RP2 mutations, and would help to develop potential therapeutics against RP in further studies.

Do not turn a blind eye to alkyl nitrite (poppers)!

Recreational users of poppers must be informed about the risk of developing poppers-associated maculopathy and the possible cumulative risk when taking poppers in combination with sildenafil. We suggest that it is an accumulative increase in cGMP which is harmful for the photoreceptor cells and may cause poppers-associated maculopathy (Fig. 1). A possible association between the use of poppers and maculopathy with visual loss was first described in 2004 (Pece et al. 2004). Poppers is the popular name for alkyl nitrite which is a short-acting, volatile drug used by inhalation. To better understand this possible association, we looked into the medical history and drug use of 10 patients with poppers-associated maculopathy. Seven patients (70%) were HIV positive, for which one patient was treated with a proteinase inhibitor at time of onset. Sixty per cent used sildenafil in combination with poppers: three were HIV positive and three were HIV negative. It may be that patients, who use poppers most, are those who get HIV and are more prone to the development of a maculopathy due to a higher cumulative dose of poppers. However, if poppers alone were a sufficient cause of maculopathy, far more women and heterosexual men would be expected among a cohort of users. Among the general population, 4% use poppers. In the homosexual community, about two per cent of the population, 30% take poppers and 6% are HIV positive (Swearingen & Klausner 2005). Hence, an additional factor like the concentration of cGMP may be the key substance damaging the photoreceptor cells in poppers-associated maculopathy. Upon oxidation, alkyl nitrites give rise to nitric oxide (NO). NO stimulates guanylate cyclase which converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). Guanylate cyclase (GC) is found in the cell body of the photoreceptor cell, that is the retinal cones (Ferrendelli & De Vries 1983). cGMP is an important substance in the photo-transduction cascade which allows Ca2+ and Na2+ to enter the cell, causing depolarization of the cell. However, very high levels of calcium will damage the photoreceptor cell (Sharma & Rohrer 2007). When patients are infected with human immunodeficiency virus (HIV), their cytokines IL-1, IL-6, interferon-gamma and TNF-alpha will increase. These cytokines activate inducible nitric oxide synthase 2 (iNOS-2), which has been shown to increase the level of NO and cGMP in the retina. In the present series, one patient was treated with a proteinase inhibitor. Proteinase inhibitors inhibit cytochrome P450 3a4. As sildenafil is broken down by cytochrome P450 3a4, proteinase inhibitors will cause an increased and prolonged concentration of sildenafil in the blood of patients using sildenafil. This indicates that also treatment of HIV patients with proteinase inhibitors might add to the development of a poppers-associated maculopathy. The three patients who were HIV negative used sildenafil besides poppers. One patient used poppers frequently for years, without any visual problems. But when he first used a combination of sildenafil and poppers, he noticed a sudden decline in visual acuity. Sildenafil, a phosphodiesterase inhibitor, prevents the degradation of cGMP. Phosphodiesterase VI is a component of the photo-transduction cascade of the photoreceptor cells. So inhibition of phosphodiesterase VI, as can be seen after the use of sildenafil, causes an increased concentration of cGMP in the photoreceptor cells. Inhibition of phosphodiesterase IV was found to induce an accumulation of cGMP with degeneration of cones in pigs (Martínez-Fernández de la Cámara et al. 2013). So, sildenafil, HIV and/or proteinase inhibitors influence a common pathway through which a rise in cGMP is induced and may thus increase the risk of developing a poppers-associated maculopathy.

714. Gene Therapy Rescues Cone Function and Viability in an Rp2 Knockout Mouse Model for X-Linked Retinitis Pigmentosa Over a Wide Dose Range and a Broad Therapeutic Time Window

Retinitis pigmentosa (RP) refers to a diverse group of inherited retinal dystrophies characterized by a progressive loss of photoreceptors, resulting in vision impairment or blindness. The prevalence of the disease is 1 in 3,500 to 4,000 worldwide. X-linked RP (XLRP) accounts for ~15% of all RP cases and is one of the most severe forms of retinal degeneration. About 10% of the total XLRP cases are caused by mutations in the RP2 gene. A recently developed Rp2 knockout (Rp2-KO) mouse model exhibits early onset, progressive cone photoreceptor dysfunction and degeneration, mimicking early cone involvement in patients with RP2 mutations. To develop gene therapy for the disease, we generated and characterized a self-complementary AAV8 vector carrying the human RP2 expression cassette (AAV8-scRK-RP2), and examined its efficacy in the Rp2-KO mice. Our initial results demonstrated successful cone function rescue following subretinal administration of the vector to the mice (ASGCT 2014, poster#122). In the present study, we continued to examine the long term safety and efficacy of the vector using a broader dose range. Four to six week-old Rp2-KO mice received unilateral subretinal injections of the vector with doses ranging from 5×107 to 1×109 vector genomes (vg)/eye. The fellow eyes were injected with vehicle as controls. Retinal function was assessed by electroretinography (ERG) and optomotor test up to 18 months of age. Our results revealed significantly higher amplitude and faster recovery of cone ERG in vector-treated eyes than control eyes in almost all dose groups through the duration of monitoring starting from 4 months of age. In addition, remarkably better visual acuity was observed in the vector-treated eyes at 18 months of age. The treatment also reversed M-opsin mis-localization, restored cone PDE expression and maintained cone viability. Interestingly, this rescue was achieved in the Rp2-KO mice even when treated at 10-month of age, suggesting the potential benefit of the treatment to older patients. Retinal toxicity was only observed in mice receiving the highest vector dose (1×109 vg/eye), as reflected by reduced rod ERG amplitude and thinner photoreceptor layer. We conclude that our RP2 AAV vector can rescue cone function and viability over a wide dose range and a broad therapeutic time window, which paves the way for future clinical studies.

Variability in Human Cone Topography Assessed by Adaptive Optics Scanning Laser Ophthalmoscopy

To assess between- and within-individual variability of macular cone topography in the eyes of young adults. Observational case series. Cone photoreceptors in 40 eyes of 20 subjects aged 19-29 years with normal maculae were imaged using a research adaptive optics scanning laser ophthalmoscope. Refractive errors ranged from -3.0 diopters (D) to 0.63 D and differed by <0.50 D in fellow eyes. Cone density was assessed on a 2-dimensional sampling grid over the central 2.4 mm × 2.4 mm. Between-individual variability was evaluated by coefficient of variation (COV). Within-individual variability was quantified by maximum difference and root mean square (RMS). Cones were cumulated over increasing eccentricity. Peak densities of foveal cones are 168 162 ± 23 529 cones/mm(2) (mean ± SD) (COV = 0.14). The number of cones within the cone-dominated foveola (0.8-0.9 mm diameter) is 38 311 ± 2319 (COV = 0.06). The RMS cone density difference between fellow eyes is 6.78%, and the maximum difference is 23.6%. Mixed-model statistical analysis found no difference in the association between eccentricity and cone density in the superior/nasal (P = .8503), superior/temporal (P = .1551), inferior/nasal (P = .8609), and inferior/temporal (P = .6662) quadrants of fellow eyes. New instrumentation imaged the smallest foveal cones, thus allowing accurate assignment of foveal centers and assessment of variability in macular cone density in a large sample of eyes. Though cone densities vary significantly in the fovea, the total numbers of foveolar cones are very similar both between and within subjects. Thus, the total number of foveolar cones may be an important measure of cone degeneration and loss.

Selective Up‐Regulation and Activation of Matrix Metalloproteinase 11 (Mmp11), Non‐Autonomous Cell Death and Pathological Dysfunctions upon Loss of Ran‐binding Protein 2 (Ranbp2).

Ranbp2 harbors functional and pathogenic idiosyncratic activities by controlling cell‐type selective, stress‐elicited or disease‐prone substrates. To probe Ranbp2 roles in neurodegeneration, we generated mice with loss of Ranbp2 expression in cone photoreceptor neurons and retinal pigment epithelium (RPE). We found that loss of Ranbp2 promotes the degeneration of the cone photoreceptors and RPE. Further, we found that degeneration of cone photoreceptors promoted the non‐autonomous death of nearby healthy rod photoreceptors. In regards to the RPE, we found that RPE degeneration led to the derangement of the cellular organization of RPE and extrusion of atrophic RPE cells to the subretinal space. Strikingly, these manifestations were followed by the breakdown of the blood‐retinal barrier that was characterized by extravasations of the nearby choriocapillaris bead. The cone and RPE degenerations were accompanied by erosion or disorganization of intracellular structures, subcellular localization of some partners of Ranbp2 and early and selective up‐regulation and activation of Mmp11, but not of any other type of matrix metalloproteinase tested. Finally, we found that functional loss of subset of Ran GTPase‐binding domains (RBDs) of Ranbp2 recapitulated the up‐regulation of Mmp11 and pathologies caused by Ranbp2 loss. Collectively, the findings support that activation of Mmp11 by Ranbp2 is under control of a subset of its RBDs and that Ranbp2‐dependent Mmp11 activation promotes the non‐autonomous death and dysfunction of healthy tissues or cells neighboring damaged cells with Ranbp2 deregulation.

Ca(2+) and Ca(2+)-interlocked membrane guanylate cyclase signal modulation of neuronal and cardiovascular signal transduction.

Ca(2+) and Ca(2+)-interlocked membrane guanylate cyclase signal modulation of neuronal and cardiovascular signal transduction.

The p110α isoform of phosphoinositide 3-kinase is essential for cone photoreceptor survival.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylates the 3'OH of the inositol ring of phosphoinositides (PIs). They are responsible for coordinating a diverse range of cellular functions. Class IA PI3K is a heterodimeric protein composed of a regulatory p85 and a catalytic p110 subunit. In this study, we conditionally deleted the p110α-subunit of PI3K in cone photoreceptor cells using the Cre-loxP system. Cone photoreceptors allow for color vision in bright light (daylight vision). Cone-specific deletion of p110α resulted in cone degeneration. Our studies suggest that PI3K signaling is essential for cone photoreceptor functions.

Cone Health and Retinoids.

Cones are photoreceptor cells used for bright light and color vision. Retinoids are vitamin A derivatives, one of which is the 11-cis aldehyde form that serves as the chromophore for both cone and rod visual pigments. In the visual disease, Type 2 Leber congenital amaurosis (LCA2), 11-cis-retinal generation is inhibited or abolished. Work by others has shown that patients with LCA2 have symptoms consistent with degenerating cones. In mouse models for LCA2, early cone degeneration is readily apparent: cone opsins and other proteins associated with the outer segment are delocalized and cell numbers decline rapidly within the first month. Rods would appear normal morphologically and functionally, if not for the absence of chromophore. Supplementation of mouse models of LCA2 with cis-retinoids has been shown to slow loss of cone photoreceptor cells if mice were maintained in darkness. Thus, 11-cis-retinal appears not only to have a role in the light response reaction but also to promote proper trafficking of the cone opsins and maintain viable cones.

Daylight vision repair by cell transplantation.

Human daylight vision depends on cone photoreceptors and their degeneration results in visual impairment and blindness as observed in several eye diseases including age-related macular degeneration, cone-rod dystrophies, or late stage retinitis pigmentosa, with no cure available. Preclinical cell replacement approaches in mouse retina have been focusing on rod dystrophies, due to the availability of sufficient donor material from the rod-dominated mouse retina, leaving the development of treatment options for cone degenerations not well studied. Thus, an abundant and traceable source for donor cone-like photoreceptors was generated by crossing neural retina leucine zipper-deficient (Nrl(-/-) ) mice with an ubiquitous green fluorescent protein (GFP) reporter line resulting in double transgenic tg(Nrl(-/-); aGFP) mice. In Nrl(-/-) retinas, all rods are converted into cone-like photoreceptors that express CD73 allowing their enrichment by CD73-based magnetic activated cell sorting prior transplantation into the subretinal space of adult wild-type, cone-only (Nrl(-/-)), or cone photoreceptor function loss 1 (Cpfl1) mice. Donor cells correctly integrated into host retinas, acquired mature photoreceptor morphology, expressed cone-specific markers, and survived for up to 6 months, with significantly increased integration rates in the cone-only Nrl(-/-) retina. Individual retinal ganglion cell recordings demonstrated the restoration of photopic responses in cone degeneration mice following transplantation suggesting, for the first time, the feasibility of daylight vision repair by cell replacement in the adult mammalian retina.

Genetic deletion of S-opsin prevents rapid cone degeneration in a mouse model of Leber congenital amaurosis.

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA), a severe hereditary blindness occurring in early childhood. The pathology is attributed to a combination of 11-cis-retinal deficiency and photoreceptor degeneration. The mistrafficking of cone membrane-associated proteins including cone opsins (M- and S-opsins), cone transducin (Gαt2), G-protein-coupled receptor kinase 1 (GRK1) and guanylate cyclase 1 (GC1) has been suggested to play a role in cone degeneration. However, their precise role in cone degeneration is unclear. Here we investigated the role of S-opsin (Opn1sw) in cone degeneration in Lrat(-) (/-), a murine model for LCA, by genetic ablation of S-opsin. We show that deletion of just one allele of S-opsin from Lrat(-) (/-) mice is sufficient to prevent the rapid cone degeneration for at least 1 month. Deletion of both alleles of S-opsin prevents cone degeneration for an extended period (at least 12 months). This genetic prevention is accompanied by a reduction of endoplasmic reticulum (ER) stress in Lrat(-) (/-) photoreceptors. Despite cone survival in Opn1sw(-/-)Lrat(-) (/-) mice, cone membrane-associated proteins (e.g. Gαt2, GRK1 and GC1) continue to have trafficking problems. Our results suggest that cone opsins are the 'culprit' linking 11-cis-retinal deficiency to cone degeneration in LCA. This result has important implications for the current gene therapy strategy that emphasizes the need for a combinatorial therapy to both improve vision and slow photoreceptor degeneration.

A mouse model for studying cone photoreceptor pathologies.

Due to the low abundance of cone photoreceptors in the mouse retina and the scarcity of alternative animal models, little is known about mechanisms of cone degeneration. Nrl knockout mice develop exclusively the cone-type of photoreceptors. However, the cone photoreceptor layer in Nrl(-/-) mice displays an irregular morphology with severe rosette formation. Retinas of Rpe65(-/-);Nrl(-/-) mice have no rosettes due to the lack of 11-cis-retinal, but also are not functional. To develop a model with a functional all-cone retina that is morphologically well structured, we generated R91W;Nrl(-/-) double-mutant mice, which express a hypomorphic Rpe65 allele (R91W). The following analyses were used to characterize the R91W;Nrl(-/-)mice: morphology by light and electron microscopy, protein distribution by immunofluorescence, cone function by electroretinography and optomotor response, RNA levels by RT-PCR, and chromophore levels by HPLC. Cone degeneration was assessed in R91W;Nrl(-/-) mice treated with MNU, and in triple R91W;Nrl(-/-);Cpfl1 and quadruple R91W;Nrl(-/-);Cpfl1;rd10 mutant mice. The all-cone retina of R91W;Nrl(-/-) mice is functional and relatively stable with only very slow age-related degeneration. Using triple and quadruple mutant mice, or a chemical treatment, we demonstrated that cone degeneration could be induced and analyzed in these mice. The reduced levels of visual chromophore prevented rosette formation and sustained function in the R91W;Nrl(-/-) retina. Thus, the R91W;Nrl(-/-) mouse allows study of the etiology of diseases related to cone degeneration in a "morphologically intact" and functional all-cone photoreceptor retina.

The progressive rod–cone degeneration (PRCD) protein is secreted through the conventional ER/Golgi-dependent pathway

Retinitis pigmentosa (RP) is the most common form of hereditary retinal degeneration. Mutations of the PRCD gene are associated with RP in both dogs and humans. To date, four distinct PRCD mutations have been reported worldwide. Here we report the clinical phenotype of another patient with PRCD mutations, carrying the known p.R18X mutation and a novel missense mutation, p.P25T. This mutation affects a highly conserved amino acid, is predicted to be damaging by several prediction tools, and was not found in the public databases or in 115 ethnically-matched control individuals. The phenotype of this patient resembles that of previously reported patients with PRCD mutations, including bull's eye maculopathy, which appears to be a hallmark of the PRCD-induced phenotype. PRCD encodes for a 54 amino acids long protein with unknown function. The first 20 amino acids appear to encode for a signal peptide (SP), suggesting that PRCD is a secreted protein. To study PRCD secretion, C-terminally myc-tagged PRCD was expressed in cultured cells. Cells and conditioned media were analyzed by Western blot. PRCD was found in both cell extracts and media. However, a truncated PRCD protein lacking the first 20 amino acids was present only in cell extracts and not in media, confirming that PRCD extracellular secretion is mediated by its N-terminal SP. To characterize the secretory pathway of PRCD, various pharmacological agents which interfere with transport of proteins through the ER and Golgi to the plasma membrane were used. PRCD secretion was significantly inhibited by all tested pharmacological agents, confirming that it is secreted through the classic ER/Golgi-dependent secretory pathway. We tested the effect of two mutations on the PRCD protein, and found that p.C2Y, but not p.P25T, affects protein stability, and that neither mutation affects secretion. Our data suggest that PRCD functions as a secreted protein. These findings shed a new light on PRCD function and the etiology of RP.

CERKL interacts with mitochondrial TRX2 and protects retinal cells from oxidative stress-induced apoptosis

Mutations in the ceramide kinase-like gene (CERKL) are associated with severe retinal degeneration. However, the exact function of the encoded protein (CERKL) remains unknown. Here we show that CERKL interacts with mitochondrial thioredoxin 2 (TRX2) and maintains TRX2 in the reduced redox state. Overexpression of CERKL protects cells from apoptosis under oxidative stress, whereas suppressing CERKL renders cells more sensitive to oxidative stress. In zebrafish, CERKL protein prominently locates in the outer segment and inner segment of the photoreceptor of the retina. Knockdown of CERKL in the zebrafish leads to an increase of retinal cell death, including cone and rod photoreceptor degeneration. Signs of oxidative damage to macromolecules were also detected in CERKL deficient zebrafish retina. Our results show that CERKL interacts with TRX2 and plays a novel key role in the regulation of the TRX2 antioxidant pathway and, for the first time, provides an explanation of how mutations in CERKL may lead to retinal cell death.

AAV-Mediated Lysophosphatidylcholine Acyltransferase 1 (Lpcat1) Gene Replacement Therapy Rescues Retinal Degeneration inrd11Mice

The retinal degeneration 11 (rd11) mouse is a newly discovered, naturally occurring animal model with early photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration. The rd11 mice carry a spontaneous mutation in the lysophosphatidylcholine acyltransferase 1 (Lpcat1) gene. Here, we evaluate whether gene replacement therapy using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can arrest retinal degeneration and restore retinal function in this model. The AAV8 (Y733F)-smCBA-Lpcat1 was delivered subretinally to postnatal day 14 (P14) rd11 mice in one eye only. At 10 weeks after injection, treated rd11 mice were examined by visually-guided behavior, electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT), and then killed for morphologic and biochemical examination. Substantial scotopic and photopic ERG signals were maintained in treated rd11 eyes, whereas untreated eyes in the same animals showed extinguished signals. The SD-OCT (in vivo) and light microscopy (in vitro) showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Almost wild-type LPCAT1 expression in photoreceptors with strong rod rhodopsin and M/S cone opsin staining, and normal visually-guided water maze behavioral performances were observed in treated rd11 mice. The results demonstrate that the tyrosine-capsid mutant AAV8 (Y733F) vector is effective for treating rapidly degenerating models of retinal degeneration and, moreover, is more therapeutically effective than AAV2 (Y444, 500, 730F) vector with the same promoter-cDNA payload. To our knowledge, this is the first demonstration of phenotypic rescue by gene therapy in an animal model of retinal degeneration caused by Lpcat1 mutation.