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AIM: To explore whether the retinal neovascularization (NV) in a genetic mutant mice model could be ameliorated in an inherited retinitis pigmentosa (RP) mouse, which would help to elucidate the possible mechanism and prevention of retinal NV diseases in clinic. METHODS: The Vldlr-/- mice, the genetic mutant mouse model of retinal NV caused by the homozygous mutation of Vldlr gene, with the rd1 mice, the inherited RP mouse caused by homozygous mutation of Pde6b gene were bred. Intercrossing of the above two mice led to the birth of the F1 hybrids, further inbreeding of which gave birth to the F2 offspring. The ocular genotypes and phenotypes of the mice from all generations were examined, with the F2 offspring grouped according to the genotypes. RESULTS: The rd1 mice exhibited the RP phenotype of outer retinal degeneration and loss of retinal function. The Vldlr-/- mice exhibited the phenotype of retinal NV obviously shown by the fundus fluorescein angiography. The F1 hydrides, with the heterozygote genotype, exhibited no phenotypes of RP or retinal NV. The F2 offspring with homozygous genotypes were grouped into four subgroups. They were the F2-I mice with the wild-type Pde6b and Vldlr genes (Pde6b+/+-Vldlr+/+), which had normal ocular phenotypes; the F2-II mice with homozygous mutant Vldlr gene (Pde6b+/+-Vldlr-/-), which exhibited the retinal NV phenotype; the F2-III mice with homozygous mutant Pde6b gene (Pde6b-/--Vldlr+/+), which exhibited the RP phenotype. Specifically, the F2-IV mice with homozygous mutant Vldlr and Pde6b gene (Pde6b-/--Vldlr-/-) showed only the RP phenotype, without the signs of retinal NV. CONCLUSION: The retinal NV can be inhibited by the RP phenotype, which implies the role of a hyperoxic state in treating retinal NV diseases.

Retinitis pigmentosa (RP) is a debilitating blinding disease affecting over 1.5 million people worldwide, but the mechanisms underlying this disease are not well understood. One of the common models used to study RP is the retinal degeneration-10 (rd10) mouse, which has a mutation in Phosphodiesterase-6b (Pde6b) that causes a phenotype mimicking the human disease. In rd10 mice, photoreceptor cell death occurs with exposure to normal light conditions, but as demonstrated in this study, rearing these mice in dark preserves their retinal function. We found that inactivating rhodopsin signaling protected photoreceptors from degeneration suggesting that the pathway activated by this G-protein-coupled receptor is causing light-induced photoreceptor cell death in rd10 mice. However, inhibition of transducin signaling did not prevent the loss of photoreceptors in rd10 mice reared under normal light conditions implying that the degeneration caused by rhodopsin signaling is not mediated through its canonical G-protein transducin. Inexplicably, loss of transducin in rd10 mice also led to photoreceptor cell death in darkness. Furthermore, we found that the rd10 mutation in Pde6b led to a reduction in the assembled PDE6αβγ2 complex, which was corroborated by our data showing mislocalization of the γ subunit. Based on our findings and previous studies, we propose a model where light activates a non-canonical pathway mediated by rhodopsin but independent of transducin that sensitizes cyclic nucleotide gated channels to cGMP and causes photoreceptor cell death. These results generate exciting possibilities for treatment of RP patients without affecting their vision or the canonical phototransduction cascade.

Purpose: The present work investigated changes in the gene expression, molecular mechanisms, and pathogenesis of inherited retinal degeneration (RD) in three different disease models, to identify predictive biomarkers for their varied phenotypes and to provide a better scientific basis for their diagnosis, treatment, and prevention.Methods: Differentially expressed genes (DEGs) between retinal tissue from RD mouse models obtained during the photoreceptor cell death peak period (Pde6brd1 at post-natal (PN) day 13, Pde6brd10 at PN23, Prphrd2 at PN29) and retinal tissue from C3H wild-type mice were identified using Illumina high-throughput RNA-sequencing. Co-expression gene modules were identified using a combination of GO and KEGG enrichment analyses and gene co-expression network analysis. CircRNA-miRNA-mRNA network interactions were studied by genome-wide circRNA screening.Results:Pde6brd1, Pde6brd10, and Prphrd2 mice had 1,926, 3,096, and 375 DEGs, respectively. Genes related to ion channels, stress, inflammatory processes, tumor necrosis factor (TNF) production, and microglial cell activation were up-regulated, while genes related to endoplasmic reticulum regulation, metabolism, and homeostasis were down-regulated. Differential expression of transcription factors and non-coding RNAs generally implicated in other human diseases was detected (e.g., glaucoma, diabetic retinopathy, and inherited retinal degeneration). CircRNA-miRNA-mRNA network analysis indicated that these factors may be involved in photoreceptor cell death. Moreover, excessive cGMP accumulation causes photoreceptor cell death, and cGMP-related genes were generally affected by different pathogenic gene mutations.Conclusion: We screened genes and pathways related to photoreceptor cell death. Additionally, up-stream regulatory factors, such as transcription factors and non-coding RNA and their interaction networks were analyzed. Furthermore, RNAs involved in RD were functionally annotated. Overall, this study lays a foundation for future studies on photoreceptor cell death mechanisms.

Prolactin (PRL) hormone functions as a pleiotropic cytokine with a protective role in the retina. We recently identified by transcriptome profiling that PRL is one of the most highly upregulated mRNAs in the retinas of mutant rcd1 (PDE6B) and xlpra2 (RPGR) dogs at advanced stages of photoreceptor disease. In the present study, we have identified the expression of a short PRL isoform that lacks exon 1 in canine retinas and analyzed the time-course of expression and localization of this isoform in the retinas of these two models. Using laser capture microdissection to isolate RNA from each of the retinal cellular layers, we found by qPCR that this short PRL isoform is expressed in photoreceptors of degenerating retinas. We confirmed by in situ hybridization that its expression is localized to the outer nuclear layer and begins shortly after the onset of disease at the time of peak photoreceptor cell death in both models. PRL protein was also detected only in mutant dog retinas. Our results call for further investigations into the role of this novel PRL isoform in retinal degeneration.

Phosphodiesterase 6B (PDE6B) is one of the most commonly mutated genes to cause autosomal recessive retinitis pigmentosa (RP), also known as rod-cone dystrophy. The PDE6B protein plays a crucial role in the phototransduction cascade. With the emerging possibility of genetic therapy for autosomal recessive PDE6B-related retinitis pigmentosa, knowledge regarding the pathogenicity and functional significance of identified PDE6B variants is crucial for genetic information for families and access to clinical trials. We collected all PDE6B variants reported in 207 autosomal recessive RP patients in publications before June 2019. The 101 unique variants obtained were classified according to the American College of Medical Genetics and Genomics guidelines. Our study provides information on the variant type, location, and predicted pathogenicity of the variants. It also highlights the need for ongoing review, as well as additional data from functional assays, to better understand the clinical significance of PDE6B variants.

Purpose The purpose of this study was to test the hypothesis that anti-oxidant and / or anti-inflammation drugs that suppress rod death in cyclic light-reared Pde6brd10 mice are also effective in dark-reared Pde6brd10 mice. Methods In untreated dark-reared Pde6brd10 mice at post-natal (P) days 23 to 24, we measured the outer nuclear layer (ONL) thickness (histology) and dark-light thickness difference in external limiting membrane-retinal pigment epithelium (ELM-RPE) (optical coherence tomography [OCT]), retina layer oxidative stress (QUEnch-assiSTed [QUEST] magnetic resonance imaging [MRI]); and microglia/macrophage-driven inflammation (immunohistology). In dark-reared P50 Pde6brd10 mice, ONL thickness was measured (OCT) in groups given normal chow or chow admixed with methylene blue (MB) + Norgestrel (anti-oxidant, anti-inflammatory), or MB or Norgestrel separately. Results P24 Pde6brd10 mice showed no significant dark-light ELM-RPE response in superior and inferior retina consistent with high cGMP levels. Norgestrel did not significantly suppress the oxidative stress of Pde6brd10 mice that is only found in superior central outer retina of males at P23. Overt rod degeneration with microglia/macrophage activation was observed but only in the far peripheral superior retina in male and female P23 Pde6brd10 mice. Significant rod protection was measured in female P50 Pde6brd10 mice given 5 mg/kg/day MB + Norgestrel diet; no significant benefit was seen with MB chow or Norgestrel chow alone, nor in similarly treated male mice. Conclusions In early rod degeneration in dark-reared Pde6brd10 mice, little evidence is found in central retina for spatial associations among biomarkers of the PDE6B mutation, oxidative stress, and rod death; neuroprotection at P50 was limited to a combination of anti-oxidant/anti-inflammation treatment in a sex-specific manner.

Purpose Disruption of proteostasis is a key event in many neurodegenerative diseases. Heat shock proteins (HSPs) participate in multiple functions associated with intracellular transport and proteostasis. We evaluated the effect of augmented HSP70 expression in mutant photoreceptors of mouse retinal degeneration models to test the hypothesis that failure to sustain HSP70 expression contributes to photoreceptor cell death. Methods We examined HSP70 expression in retinas of wild-type and mutant mice by RNA and protein analysis. A transgenic mouse line, TgCrx-Hspa1a-Flag, was generated to express FLAG-tagged full-length HSP70 protein under control of a 2.3 kb mouse Crx promoter. This line was crossed to three distinct retinal degeneration mouse models. Retinal structure and function were evaluated by histology, immunohistochemistry, and electroretinography. Results In seven different mouse models of retinal degeneration, we detected transient elevation of endogenous HSP70 expression at early stages, followed by a dramatic reduction as cell death ensues, suggesting an initial adaptive response to cellular stress. Augmented expression of HSP70 in RHOT17M mice, in which mutant rhodopsin is misfolded, marginally improved photoreceptor survival, whereas elevated HSP70 led to more severe retinal degeneration in rd10 mutants that produce a partially functional PDE6B. In Rpgrip1-/- mice that display a ciliary defect, higher HSP70 had no impact on photoreceptor survival or function. Conclusions HSP70 overexpression has divergent effects in photoreceptors determined, at least in part, by the nature of the mutant protein each model carries. Additional investigations on HSP pathways and associated chaperone networks in photoreceptors are needed before designing therapeutic strategies targeting proteostasis.

Objective To identify the underlying mutation in a recently identified early-onset progressive retinal atrophy (PRA) in the Spanish Water Dog (SWD) breed. Animal studied Eighteen SWDs were used in this study. Six SWDs diagnosed with PRA and 12 phenotypically normal SWDs. Procedures An exclusion analysis using an established microsatellite panel to screen PRA candidate genes was combined with whole genome sequencing of two affected SWD siblings and two phenotypically normal SWDs (a sibling and the dam). Results A 6-bp deletion was identified in exon 19 of PDE6B removing two highly conserved amino acids from the enzymatic domain of the PDE6B protein (c.2218-2223del; p.Phe740_Phe741del). This segregated with the disease status in the small study pedigree. Conclusions Identification of this novel PDE6B mutation adds to the already described PDE6B mutations responsible for PRA in the Irish Setter, Sloughi, and American Staffordshire Terrier dog breeds. A DNA-based test was designed to allow breeders to genotype their animals and make informed breeding decisions in the effort to eradicate PRA from the SWD breed.

Photoreceptor loss is the final common endpoint in most retinopathies that lead to irreversible blindness, and there are no effective treatments to restore vision1,2. Chemical reprogramming of fibroblasts offers an opportunity to reverse vision loss; however, the generation of sensory neuronal subtypes such as photoreceptors remains a challenge. Here we report that the administration of a set of five small molecules can chemically induce the transformation of fibroblasts into rod photoreceptor-like cells. The transplantation of these chemically induced photoreceptor-like cells (CiPCs) into the subretinal space of rod degeneration mice (homozygous for rd1, also known as Pde6b) leads to partial restoration of the pupil reflex and visual function. We show that mitonuclear communication is a key determining factor for the reprogramming of fibroblasts into CiPCs. Specifically, treatment with these five compounds leads to the translocation of AXIN2 to the mitochondria, which results in the production of reactive oxygen species, the activation of NF-κB and the upregulation of Ascl1. We anticipate that CiPCs could have therapeutic potential for restoring vision. A set of five small molecules can induce the transformation of fibroblasts into rod photoreceptor-like cells, which can partially restore pupil reflex and visual function when transplanted into a rod degeneration mouse model.

An HDR-based Cas9/RecA system was developed to precisely correct the Pde6b mutation in rods of rd1 mice to restore their vision., Although Cas9-mediated genome editing has been widely used to engineer alleles in animal models of human inherited diseases, very few homology-directed repair (HDR)–based genetic editing systems have been established in postnatal mouse models for effective and lasting phenotypic rescue. Here, we developed an HDR-based Cas9/RecA system to precisely correct Pde6b mutation with increased HDR efficiency in postnatal rodless (rd1) mice, a retinitis pigmentosa (RP) mutant model characterized by photoreceptor degeneration and loss of vision. The Cas9/RecA system incorporated Cas9 endonuclease enzyme to generate double-strand breaks (DSBs) and bacterial recombinase A (RecA) to increase homologous recombination. Our data revealed that Cas9/RecA treatment significantly promoted the survival of both rod and cone photoreceptors, restored the expression of PDE6B in rod photoreceptors, and enhanced the visual functions of rd1 mice. Thus, this study provides a precise therapeutic strategy for RP and other genetic diseases.

The retinal degeneration model rd10 contains a missense mutation of the catalytic PDE6 β subunit, which hydrolyzes cGMP in response to light. This model produces cell death more slowly than others caused by PDE6 loss of function, making it of particular interest for studying potential therapeutics. We used morphology, biochemistry, and single-cell physiology to examine the mechanism of rd10 degeneration. Our results show that the mutation produces no alteration of Pde6b RNA but does dramatically decrease maximal and basal PDE6 activity, apparently caused by a decrease in protein stability and transport. The enzymatic properties of the remaining mutant PDE6 appear to be nearly normal. We demonstrate that an increase in free cGMP, which would result from decreased PDE6 activity and serve to increase opening of the cGMP-gated channels and calcium influx, is an underlying cause of cell death: degeneration of rd10/Cngb1(−/−) double mutants is slower than the parent rd10 line. Paradoxically, degeneration in rd10/Cngb1(−/−) is also slower than in Cngb1(−/−). This rescue is correlated with a lowering of cGMP content in Cngb1(−/−) retinas and suggests that it may be caused by mislocalization of active PDE6. Single-cell recordings from rd10 rods show that the rates of rise and decay of the response are significantly slower; simulations indicate that these changes are primarily the result of the decrease in PDE6 concentration and rod collecting area. Together, these results provide insights into the complex mechanisms that underlie rd10-mediated retinal degeneration and a cautionary note for analysis of therapeutic interventions.

Background Retinitis pigmentosa (RP) is a group of hereditary retinal neurodegenerative conditions characterized by primary dysfunction and death of photoreceptor cells, resulting in visual loss and, eventually, blindness. To date, no effective therapies have been transferred to clinic. Given the diverse genetic etiology of RP, targeting common cellular and molecular retinal alterations has emerged as a potential therapeutic strategy. Methods Using the Pde6b rd10/rd10 mouse model of RP, we investigated the effects of daily intraperitoneal administration of VP3.15, a small-molecule heterocyclic GSK-3 inhibitor. Gene expression was analyzed by quantitative PCR and protein expression and phosphorylation by Western blot. Photoreceptor preservation was evaluated by histological analysis and visual function was assessed by electroretinography. Results In rd10 retinas, increased expression of pro-inflammatory markers and reactive gliosis coincided with the early stages of retinal degeneration. Compared with wild-type controls, GSK-3β expression (mRNA and protein) remained unchanged during the retinal degeneration period. However, levels of GSK-3βSer9 and its regulator AktSer473 were increased in rd10 versus wild-type retinas. In vivo administration of VP3.15 reduced photoreceptor cell loss and preserved visual function. This neuroprotective effect was accompanied by a decrease in the expression of neuroinflammatory markers. Conclusions These results provide proof of concept of the therapeutic potential of VP3.15 for the treatment of retinal neurodegenerative conditions in general, and RP in particular., We thank Cayetana Murillo, Laura Ramírez and the staff of the CIB animal house and microscopy facilities for technical support. We thank Violeta Gómez-Vicente for advice on GSK-3 immunostaining. We thank Dr. GarcíaPardo and Dr. Labandeira-García for providing the pNF-κBser536 antibody and the N9 cell line, respectively. ASC and JZD are recipients of UCM and MECD (FPU13-00362) predoctoral fellowships, respectively, This work was supported by grants from the Spanish MINECO (SAF2012- 37979-C03-01 and SAF2016-76639-R to AM, SAF2013-41059-R and SAF2016- 75681-R to EJdlR, and PI13-02098 to PdlV) and Instituto de Salud Carlos III and cofinanced by FEDER (PI17/01601 and RETICS RD16/0019/0009 to IL).

Background/Aims: Our laboratory discovered a Kunming mouse with enormous electroretinogram (ERG) defects. Its auditory brainstem response (ABR) threshold was significantly elevated and closely resembled the features of Usher syndrome (USH). This study sought to cross these USH-like mice (named KMush/ush mice) with CBA/CaJ mice to establish recombinant inbred strains and identify their phenotypes and genotypes. Methods: KMush/ush mice were crossed with CBA/CaJ mice to establish inbred strains by sibling mating. ERG, ABR, ocular fundus morphology, histological examinations of the retina and inner ear, quantitative real-time polymerase chain reaction, western blotting, and exon sequencing were performed to assess the phenotypes and genotypes of the offspring strains. Results: The F1 hybrids from crossing KMush/ush and CBA/CaJ mice had normal ERG and ABR responses. The F2 offspring from intercrossing the F1 mice showed a segregation of the retinitis pigmentosa (RP) and hearing loss phenotypes. The CBA-1ush/ush mice had an RP phenotype that was characterized by a vanished ERG waveform and loss of the outer nuclear layer. Their Pde6b gene had a nonsense mutation that resulted in the failure of protein production in western blotting. However, the ABR threshold of this strain of mice was normal. The CBA-2ush/ush mice had normal retinal function and architecture. Their ABR threshold was increased, with a dramatic degeneration of the stereocilia bundles in the outer hair cells of the inner ear. Whole exome sequencing and exon sequencing revealed a deletion of one base pair in exon 31 of the Adgrv1 gene, which would result in the premature termination of protein encoding. The level of Adgrv1 mRNA was reduced in the CBA-2ush/ush mice. The CBA-3ush/ush mice had phenotypes of RP, elevated ABR threshold, and degeneration of the stereocilia bundles in the outer hair cells. They were closely associated with the nonsense mutations of Pde6b and Adgrv1, respectively. Conclusion: We isolated a mouse strain with hearing loss from inbred mice with retinal degeneration and established it as a recombinant inbred strain with a spontaneous mutation in Adgrv1, the human Usher syndrome 2C gene. The retinal degeneration was cause by a mutation in Pde6b, while the hearing loss was caused by a mutation in Adgrv1.

Aryl-hydrocarbon receptor interacting protein-like 1 (AIPL1) is essential to stabilize cGMP phosphodiesterase 6 (PDE6) in rod photoreceptors. Mutation of AIPL1 leads to loss of PDE6, accumulation of intracellular cGMP, and rapid degeneration of rods. To understand the metabolic basis for the photoreceptor degeneration caused by excessive cGMP, we performed proteomics and phosphoproteomics analyses on retinas from AIPL1-/- mice at the onset of rod cell death. AIPL1-/- retinas have about 18 times less than normal PDE6a and no detectable PDE6b. We identified twelve other proteins and thirty-nine phosphorylated proteins related to cell metabolism that are significantly altered preceding the massive degeneration of rods. They include transporters, kinases, phosphatases, transferases, and proteins involved in mitochondrial bioenergetics and metabolism of glucose, lipids, amino acids, nucleotides, and RNA. In AIPLI-/- retinas mTOR and proteins involved in mitochondrial energy production and lipid synthesis are more dephosphorylated, but glycolysis proteins and proteins involved in leucine catabolism are more phosphorylated than in normal retinas. Our findings indicate that elevating cGMP rewires cellular metabolism prior to photoreceptor degeneration and that targeting metabolism may be a productive strategy to prevent or slow retinal degeneration.

Zebrafish are an excellent animal model for research on vertebrate development and human diseases. Sophisticated genetic tools including large-scale mutagenesis methodology make zebrafish useful for studying neuronal degenerative diseases. Here, we review zebrafish models of inherited ophthalmic diseases, focusing on cGMP metabolism in photoreceptors. cGMP is the second messenger of phototransduction, and abnormal cGMP levels are associated with photoreceptor death. cGMP concentration represents a balance between cGMP phosphodiesterase 6 (PDE6) and guanylate cyclase (GC) activities in photoreceptors. Various zebrafish cGMP metabolism mutants were used to clarify molecular mechanisms by which dysfunctions in this pathway trigger photoreceptor degeneration. Here, we review the history of research on the retinal degeneration (rd) mutant mouse, which carries a genetic mutation of PDE6b, and we also highlight recent research in photoreceptor degeneration using zebrafish models. Several recent discoveries that provide insight into cGMP toxicity in photoreceptors are discussed.

Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c(w59) mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c(w59) embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c(w59) mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.

Massive parallel sequencing enables identification of numerous genetic variants in mutant organisms, but determining pathogenicity of any one mutation can be daunting. The most commonly studied preclinical model of retinitis pigmentosa called the "rodless" (rd1) mouse is homozygous for two mutations: a nonsense point mutation (Y347X) and an intronic insertion of a leukemia virus (Xmv-28). Distinguishing which mutation causes retinal degeneration is still under debate nearly a century after the discovery of this model organism. Here, we performed gene editing using the CRISPR/Cas9 system and demonstrated that the Y347X mutation is the causative variant of disease. Genome editing in the first generation produced animals that were mosaic for the corrected allele but still showed neurofunction preservation despite low repair frequencies. Furthermore, second-generation CRISPR-repaired mice showed an even more robust rescue and amelioration of the disease. This predicts excellent outcomes for gene editing in diseased human tissue, as Pde6b, the mutated gene in rd1 mice, has an orthologous intron-exon relationship comparable with the human PDE6B gene. Not only do these findings resolve the debate surrounding the source of neurodegeneration in the rd1 model, but they also provide the first example of homology-directed recombination-mediated gene correction in the visual system.

9 p.-6 fig., PURPOSE. The induction of proinsulin expression by transgenesis or intramuscular gene therapy has been shown previously to retard retinal degeneration in mouse and rat models of retinitis pigmentosa (RP), a group of inherited conditions that result in visual impairment. We investigated whether intraocular treatment with biodegradable poly (lactic-co-glycolic) acid microspheres (PLGA-MS) loaded with proinsulin has cellular and functional neuroprotective effects in the retina, METHODS. Experiments were performed using the Pde6brd10 mouse model of RP. Methionylated human recombinant proinsulin (hPI) was formulated in PLGA-MS, which were administered by intravitreal injection on postnatal days (P) 14 to 15. Retinal neuroprotection was assessed at P25 by electroretinography, and by evaluating outer nuclear layer (ONL) cellular preservation. The attenuation of photoreceptor cell death by hPI was determined by TUNEL assay in cultured P22 retinas, as well as Akt phosphorylation by immunoblotting, RESULTS. We successfully formulated hPI PLGA-MS to deliver the active molecule for several weeks in vitro. The amplitude of b-cone and mixed b-waves in electroretinographic recording was significantly higher in eyes injected with hPI-PLGA-MS compared to control eyes.Treatment with hPI-PLGA-MS attenuated photoreceptor cell loss, as revealed by comparing ONL thickness and the number of cell rows in this layer in treated versus untreated retinas. Finally, hPI prevented photoreceptor cell death and increased AktThr308 phosphorylation in organotypic cultured retinas., CONCLUSIONS. Retinal degeneration in the rd10 mouse was slowed by a single intravitreal injection of hPI-PLGA-MS. Human recombinant proinsulin elicited a rapid and effective neuroprotective effect when administered in biodegradable microspheres, which may constitute a future potentially feasible delivery method for proinsulin-based treatment of RP., Supported by Grants from the Spanish Ministerio de Ciencia e Innovacion (MICINN) and Spanish Ministerio de EconomÍa y Competitividad (MINECO), SAF2010-21879 (EJdlR and PdlV), SAF2013-41059-R (FdP and EJdlR), and technical personnel support from CIBERDEM, ISCIII, Madrid, Spain

Retinitis pigmentosa (RP) comprises a group of inherited retinal conditions that leads to visual field loss and retinal degeneration (RD). The most common feature of all forms of RP is a gradual degeneration of photoreceptor cells: rods and cones. The rods and cones are the cells responsible for converting light into electrical impulses that are transmitted to the brain where “seeing” actually occurs. RP is a common form of RD. Many animal models of RD are available and studying them has led to a better understanding of the pathology of the disease, and to the development of therapeutic strategies aimed at curing or slowing down RD. This chapter will focus on mouse models of RD and specifically, the use of the rd10 (Pde6b rd10 ) model is an example of how an RD model can be used to explore RP. Mouse models, with their well-developed genetics and similarity to human physiology and anatomy, serve as powerful tools to investigate the etiology of human RD. Mutant mice also provide reproducible, experimental systems for elucidating pathways of normal development and function. Through using these mouse models much progress has been made in elucidating gene defects underlying retinal diseases, understanding of mechanisms leading to disease, and in designing strategies and drugs for translational research and gene-based therapies to delay the progression of disease.

Genes encoding the proteins of cytoskeletal intermediate filaments (IF) are tightly regulated, and they are important for establishing neural connections. However, it remains uncertain to what extent neurological disease alters IF gene expression or impacts cells that express IFs. In this study, we determined the onset of visual deficits in a mouse model of progressive retinal degeneration (Pde6b(-) mice; Pde6b(+) mice have normal vision) by observing murine responses to a visual task throughout development, from postnatal day (PND) 21 to adult (N=174 reliable observations). Using Q-PCR, we evaluated whether expression of the genes encoding two Type III IF proteins, glial fibrillary acidic protein (GFAP) and vimentin was altered in the visual cortex before, during, and after the onset of visual deficits. Using immunohistochemical techniques, we investigated the impact of vision loss on the density and morphology of astrocytes that expressed GFAP and vimentin in the visual cortex. We found that Pde6b(-) mice displayed 1) evidence of blindness at PND 49, with visual deficits detected at PND 35, 2) reduced GFAP mRNA expression in the visual cortex between PND 28 and PND 49, and 3) an increased ratio of vimentin:GFAP-labeled astrocytes at PND 49 with reduced GFAP cell body area. Together, these findings demonstrate that retinal degeneration modifies cellular and molecular indices of glial plasticity in a visual system with drastically reduced visual input. The functional consequences of these structural changes remain uncertain.

Retinal degenerations such as Retinitis Pigmentosa remain difficult to treat given the diverse array of genes responsible for their aetiology. Rather than concentrate on specific genes, our focus is on identifying therapeutic avenues for the treatment of retinal disease that target general survival mechanisms or pathways. Norgestrel is a synthetic progestin commonly used in hormonal contraception. Here, we report a novel anti-apoptotic role for Norgestrel in diseased mouse retinas in vivo. Dosing with Norgestrel protects photoreceptor cells from undergoing apoptosis in two distinct models of retinal degeneration; the light damage model and the Pde6b(rd10) model. Photoreceptor rescue was assessed by analysis of cell number, structural integrity and function. Improvements in cell survival of up to 70% were achieved in both disease models, indicating that apoptosis had been halted or at least delayed. A speculative mechanism of action for Norgestrel involves activation of survival pathways in the retina. Indeed, Norgestrel increases the expression of basic fibroblast growth factor which is known to both promote cell survival and inhibit apoptosis. In summary, our results demonstrate significant protection of photoreceptor cells which may be attributed to Norgestrel mediated activation of endogenous survival pathways within the retina.

In contrast to most mammalian species, the ciliary processes in the mouse eye form an irregular pattern. Different strains were studied using scanning electron microscopy. The ciliary processes of C57BL/6J animals showed quadrant-specific characteristics: in the superior quadrant, large and radial oriented processes were present. In the inferior quadrant, the processes were small but still mainly radial oriented. In the temporal quadrant, the processes showed a radial, longitudinal course, some being L-shaped. In the nasal quadrant, few processes were oriented longitudinal. In DBA/2 animals, the processes were shorter and the radial orientation less developed. NMRI animals showed the shortest processes with no increase towards the superior quadrant. Additionally we investigated age-related changes in the ciliary processes of Pde6b(rd1) mice, which develop retinal degeneration. In C57BL/6J mice, the ciliary body shape, size and architecture was comparable between 3 and 10 months of age, but showed a mild shortening of the pars plicata in the temporal, inferior and nasal quadrants in animals older than 20 months of age. The parameters of the ciliary body in 3 months old Pde6b(rd1) mice were comparable to those of age-matched C57BL/6J mice. Pde6b(rd1) mice 10 months old revealed significant shortening of the total width of the ciliary body and of the length of ciliary processes in all quadrants. The shape and architecture of the ciliary processes remained preserved.

We report the chromosomal localization, mutant gene identification, ophthalmic appearance, histology, and functional analysis of two new hereditary mouse models of retinal degeneration not having the Pde6brd1("r", "rd", or "rodless") mutation. One strain harbors an autosomal recessive mutation that maps to mouse chromosome 5. Sequence analysis showed that the retinal degeneration is caused by a missense point mutation in exon 13 of the beta-subunit of the rod cGMP phosphodiesterase (beta-PDE) gene (Pde6b). The gene symbol for this strain was set as Pde6brd10, abbreviated rd10 hereafter. Mice homozygous for the rd10 mutation showed histological changes at postnatal day 16 (P16) of age and sclerotic retinal vessels at four weeks of age, consistent with retinal degeneration. Retinal sections were highly positive for TUNEL and activated caspase-3 immunoreactivity, specifically in the outer nuclear layer (ONL). ERGs were never normal, but rod and cone ERG a- and b-waves were easily measured at P18 and steadily declined over 90% by two months of age. Protein extracts from rd10 retinas were positive for beta-PDE immunoreactivity starting at about the same time as wild-type (P10), though signal averaged less than 40% of wild-type. Interestingly, rearing rd10 mice in total darkness delayed degeneration for at least a week, after which morphological and functional loss progressed irregularly. With the second strain, a complementation test with rd1 mice revealed that the retinal degeneration phenotype observed represents a possible new allele of Pde6b. Sequencing demonstrated a missense point mutation in exon 16 of the beta-subunit of rod phosphodiesterase gene, different from the point mutations in rd1 and rd10. The gene symbol for this strain was set as Pde6bnmf137, abbreviated nmf137 hereafter. Mice homozygous for this mutation showed retinal degeneration with a mottled retina and white retinal vessels at three weeks of age. The exon 13 missense mutation (rd10) is the first known occurrence of a second mutant allele spontaneously arising in the Pde6b gene in mice and may provide a model for studying the pathogenesis of autosomal recessive retinitis pigmentosa (arRP) in humans. It may also provide a better model for experimental pharmaceutical-based therapy for RP because of its later onset and milder retinal degeneration than rd1 and nmf137.

Usher syndrome is a group of autosomal recessive diseases characterized by congenital deafness and retinitis pigmentosa. In a mouse model for Usher syndrome, KMush/ush, discovered in our laboratory, we measured the phenotypes, characterized the architecture and morphology of the retina, and quantified the level of expression of pde6b and ush2a between postnatal (P) days 7, and 56. Electroretinograms and auditory brainstem response were used to measure visual and auditory phenotypes. Fundus photography and light microscopy were used to measure the architecture and morphology of the retina. Quantitative real-time PCR was used to measure the expression levels of mRNA. KMush/ush mice had low amplitudes and no obvious waveforms of Electroretinograms after P14 compared with controls. Thresholds of auditory brainstem response in our model were higher than those of controls after P14. By P21, the retinal vessels of KMush/ush mice were attenuated and their optic discs had a waxy pallor. The retinas of KMush/ush mice atrophied and the choroidal vessels were clearly visible. Notably, the architecture of each retinal layer was not different as compared with control mice at P7, while the outer nuclear layer (ONL) and other retinal layers of KMush/ush mice were attenuated significantly between P14 and P21. ONL cells were barely seen in KMush/ush mice at P56. As compared with control mice, the expression of pde6b and ush2a in KMush/ush mice declined significantly after P7. This study is a first step toward characterizing the progression of disease in our mouse model. Future studies using this model may provide insights about the etiology of the disease and the relationships between genotypes and phenotypes providing a valuable resource that could contribute to the foundation of knowledge necessary to develop therapies to prevent the retinal degeneration in patients with Usher Syndrome.

While photoreceptor loss is the most devastating result of inherited retinal degenerations such as retinitis pigmentosa, inner retinal neurons also undergo significant alteration. Detailing these changes has become important as many vision restorative therapies target the remaining neurons. In this study, the rd1-Fos-Tau-LacZ (rd1-FTL) mouse model was used to explore inner retinal change at a late stage of retinal degeneration, after the loss of photoreceptor nuclei. The rd1-FTL model carries a mutation in the phosphodiesterase gene, Pde6b, and an axonally targeted transgenic beta galactosidase reporter system under the control of the c-fos promoter. Retinae of transgenic rd1-FTL mice and control FTL animals aged 2–12 months were processed for indirect fluorescence immunocytochemistry. At 2 months of age, a time when the majority of photoreceptor nuclei are lost, there was negligible c-fos reporter (FTL) expression, however, from 4 months, reporter expression was observed to increase within subpopulations of amacrine and ganglion cells within the central retina. These areas of inner retinal FTL expression coincided with regions that contained aberrant Müller cells. Specifically, these cells exhibited reduced glutamine synthetase and Kir4.1 immunolabelling, whilst showing evidence of proliferative gliosis (increased cyclinD1 and glial fibrillary acidic protein expression). These changes were limited to distinct regions where cone photoreceptor terminals were absent. Overall, these results highlight that distinct areas of the rd1-FTL central retina undergo significant glial alterations after cone photoreceptor loss. These areas coincide with up-regulation of the c-fos reporter in the inner retina, which may represent a change in neuronal function/plasticity. The rd1-FTL mouse is a useful model system to probe changes that occur in the inner retina at later stages of retinal degeneration.

Many of the inbred mouse strains commonly used in biomedical research are homozygous for the rd1 mutation of the Pde6b gene, which causes retinal degeneration. To dissociate the behavioural effects of rd1 homozygosity from those of the genetic background of the host strain in the most widely used paradigms for evaluating the cognitive abilities of mice, two rd1 homozygous strains (C3H/HeJ and CBA/J) were compared with two Pde6b wild-type strains, each possessing a genetic background identical (C3A.BLiA-Pde6b+/J) or very similar (CBA/CaJ) to that of its rd1 homozygous relative. In the fear conditioning procedure, the presence of the rd1 mutation had no effect on performance at any stage, as the superior contextual learning of the CBA/J and CBA/CaJ strains could be explained by genetic background effects alone. In the Morris water maze, only the Pde6b wild-type C3A.BLiA-Pde6b+/J and CBA/CaJ strains were able to demonstrate spatial learning. The study thus demonstrates how retinal degeneration and genetic background have different effects in these two tests of hippocampus-dependent learning and memory.

We have generated a large number of potential eye and vision mutants by a mouse mutagenesis programme and sought to develop a rapid method for testing vision. This experiment assesses whether the visual tracking drum can differentiate between mice with and without retinal degeneration. The population of mice tested had a proportion with retinal degeneration, the remainder having normal retinas. Mice were observed in the visual tracking drum for a head tracking response to a visual stimulus. They were then examined for retinal degeneration. Of 204 animals tested, 138 had retinal degeneration. None of these showed any head tracking response. Of the remaining 66 with normal retinas, 45 showed a normal response, and 19 of the 21 mice which did not respond on a first test showed a normal response when the test was repeated. The visual tracking drum differentiates reliably between mice with normal vision and mice with severely decreased vision. There was a relatively high rate of non-responders among the normal sighted mice on a first test, but the test is rapid enough to be repeated easily.

Mutations in USH2A are a common cause of Retinitis Pigmentosa (RP). Among the most frequently reported USH2A variants, c.2276G>T (p.C759F) has been found in both affected and healthy individuals. The pathogenicity of this variant remains controversial since it was detected in homozygosity in two healthy siblings of a Spanish family (S23), eleven years ago. The fact that these individuals remain asymptomatic today, prompted us to study the presence of other pathogenic variants in this family using targeted resequencing of 26 retinal genes in one of the affected individuals. This approach allowed us to identify one novel pathogenic homozygous mutation in exon 13 of PDE6B (c.1678C>T; p.R560C). This variant cosegregated with the disease and was absent in 200 control individuals. Remarkably, the identified variant in PDE6B corresponds to the mutation responsible of the retinal degeneration in the naturally occurring rd10 mutant mice. To our knowledge, this is the first report of the identification of the rd10 mice mutation in a RP family. These findings, together with a review of the literature, support the hypothesis that homozygous p.C759F mutations are not pathogenic and led us to exclude the implication of p.C759F in the RP of family S23. Our results indicate the need of re-evaluating all families genetically diagnosed with this mutation.

Early retinal degeneration (erd) is an early onset progressive retinal atrophy, a hereditary canine retinal disease phenotypically similar to human retinitis pigmentosa (RP). In previous efforts to identify the erd locus, canine homologs of genes causally associated with RP in humans, such as opsin (RHO), the β-subunit gene for cyclic GMP phosphodiesterase (PDE6B), and RDS/peripherin, were excluded. A genome-wide screen was undertaken on canine families segregating the erd disease. Analysis of over 150 canine-specific markers has localized erd to a single linkage group comprising two previously identified canine linkage groups, 20 and 26, corresponding to canine radiation hybrid groups RH.34-a and RH.40-a. Multipoint analysis places erd in the interval between marker FH2289 (distance 23.6 cM) and FH2407 (5.9 cM) with a lod score of 12.23. Although the erd linkage group has not been assigned to an identified canine chromosome, conserved synteny of this linkage group with human 12p13–q13 suggests several candidates for erd and identifies a novel retinal degeneration locus. The rapid progress now occurring in canine genetics will expedite identification of the genes and molecular mechanisms underlying the inherited traits and diseases that make the dog a unique asset for study of mammalian traits.

Rod-cone dysplasia 1 (rcd1) in Irish setters is caused by a nonsense mutation in the cGMP phosphodiesterase beta-subunit gene (PDE6B). We examined the frequency of the mutant allele in the Irish setter population and determined if the defect is present in dogs of other breeds which are affected with other inherited photoreceptor diseases. Between 1994 and 1997, samples were obtained from 436 clinically normal Irish setters, a red wolf, and dogs from 23 different breeds. The mutation in codon 807 of PDE6B was detected in genomic DNA by heteroduplex analysis, allele-specific PCR, or restriction enzyme digestion. Of the 436 samples from clinically normal setters, 34 contained the mutation in one of the two PDE6B alleles (carrier rate = 7.8%). In contrast, the same mutation was not found in the red wolf or dogs of other breeds affected with PRA or inherited photoreceptor diseases. The high percentage of tested carriers, however, is not representative of the number of carriers in the population since some dogs tested were closely related and did not represent a random sample of the Irish setter breed.

Purpose To identify the causative mutations in two early-onset canine retinal degenerations, crd1 and crd2, segregating in the American Staffordshire terrier and the Pit Bull Terrier breeds, respectively. Methods Retinal morphology of crd1- and crd2-affected dogs was evaluated by light microscopy. DNA was extracted from affected and related unaffected controls. Association analysis was undertaken using the Illumina Canine SNP array and PLINK (crd1 study), or the Affymetrix Version 2 Canine array, the "MAGIC" genotype algorithm, and Fisher's Exact test for association (crd2 study). Positional candidate genes were evaluated for each disease. Results Structural photoreceptor abnormalities were observed in crd1-affected dogs as young as 11-weeks old. Rod and cone inner segment (IS) and outer segments (OS) were abnormal in size, shape, and number. In crd2-affected dogs, rod and cone IS and OS were abnormal as early as 3 weeks of age, progressing with age to severe loss of the OS, and thinning of the outer nuclear layer (ONL) by 12 weeks of age. Genome-wide association study (GWAS) identified association at the telomeric end of CFA3 in crd1-affected dogs and on CFA33 in crd2-affected dogs. Candidate gene evaluation identified a three bases deletion in exon 21 of PDE6B in crd1-affected dogs, and a cytosine insertion in exon 10 of IQCB1 in crd2-affected dogs. Conclusions Identification of the mutations responsible for these two early-onset retinal degenerations provides new large animal models for comparative disease studies and evaluation of potential therapeutic approaches for the homologous human diseases.

Retinitis pigmentosa (RP) is a well known inherited retinal dystrophy that leads to progressive loss of vision, and eventually complete blindness due to photoreceptor cell death. RP is restricted to the eye, specifically the retina and photoreceptors. Its prevalence is estimated to be at 1/4000 (1–3). RP is caused by progressive loss of rods followed by the loss of cones. Affected individuals start experiencing night blindness in the first two decades of life. Peripheral visual field constriction leads to ‘tunnel vision’, progressive loss of visual acuity (VA) leads to complete loss of all vision (4). On fundus exam, RP is classically characterized by retinal atrophy, optic nerve head pallor, vascular narrowing and a variety of peripheral retinal pigmentary deposits including bone spicules. RP can be restricted to the eye (non-syndromic) or a retinal degeneration indistinguishable from RP can be associated with a syndrome. The syndromic features can be very obvious or very subtle. Also, the syndromic features can be present at the time of diagnosing the retinal degeneration, or they may appear much later. Examples are Bardet–Biedl syndrome (BBS), Usher Syndrome (USH) and Senior-Loken Syndrome (SLS). The mode of inheritance of RP may be autosomal dominant [autosomal dominant retinitis pigmentosa (adRP); 5–25%], autosomal recessive [autosomal recessive retinitis pigmentosa (arRP); 5–20%], rarely digenic, mitochondrial or X-linked [Xlinked retinitis pigmentosa (xlRP); 5–15%] (5). There are currently 36 genes implicated in arRP that only explain ~50% of the cases (RetNet), illustrating that many mutations and new genes still need to be discovered. RPE65, which is expressed in the retinal pigment epithelium (RPE), PDE6A and PDE6B, which are both phosphodiesterase subunits in the phototransduction cascade, EYS and USH2A, are among the most well-known or common genetic etiologies (5, 6). Interestingly, several genes associated with RP (in patients without systemic disease) can also be mutated in syndromic disorders such as BBS1 and USH2 (7, 8) and SLS which is an autosomal recessive disease affecting primarily the kidney and retina, leading to nephronophthisis (NPHP) and RP, respectively (9). Of particular interest in RP and these syndromes is the emergence of a group of genes and proteins that function in and are localized to the cilium, the so-called ciliary genes. In photoreceptor outer segments (OS, which represent modified cilia), the stacked discs house the proteins for visual function with several important RP ciliary gene products, e.g. RPGR, RPGRIP1, USH2A, CEP290, LCA5, and TULP1. We identified an atypical form of arRP in a French-Canadian family (Fig. 1) and excluded mutations in all the known arRP genes. We hypothesized that this family carries mutations in a novel RP specific gene and subsequently identified homozygous mutations in WDR19. At the time of discovery (August 2011), there was no published article in the literature linking this gene to any human disease and specifically to arRP. On re-evaluation of this family, we discovered that several of the RP patients had sub-clinical renal cysts, which prompted us to hypothesize that this gene may be mutated in RP patients with frank kidney failure, a disorder coined SLS. We then performed mutation analysis in this gene in a cohort of patients with RP in Quebec and a cohort with the syndromic form of RP, namely SLS and identified more RP families and several SLS families with mutations. At this time, both Bredrup et al. in their original article and Huber et al., in their review article, described the involvement of WDR19, a ciliary gene, in a variety of ciliopathies including Sensenbrenner, Jeune syndrome and isolated NPHP (10, 11). Similarly, Halbritter et al. reported the first case of Caroli disease, which is a rare inherited disorder most commonly characterized by congenital cystic dilatation of the intrahepatic biliary ducts secondary to ectasia, caused by mutations in WDR19. However, there is yet no study confirming clearly the involvement of WDR19 in arRP as well as in SLS (12). Fig. 1 (a) Family A pedigree. (b) Retinal photo showing an atrophic maculopathy and severe narrowing of the blood vessels and temporal pallor of the optic nerve head. (c) Fundus autofluorescence showing the ‘bear claw’ maculopathy and hypo-fluorescence ... In the process of identifying a novel gene for RP and SLS, we also identified a novel genotype–phenotype correlation in the arRP families. We illustrate that the combined use of Arrayed Primer Extension (APEX) technology, homozygosity mapping by single nucleotide polymorphism (SNP) genotyping and next generation sequencing (NGS), can rapidly and reliably lead to new gene discovery.

Mutations in the gene encoding the beta subunit of rod cGMP phosphodiesterase are known causes of photoreceptor degeneration in two animal models of retinitis pigmentosa, the rd (retinal degeneration) mouse and the Irish setter dog with rod/cone dysplasia. Here we report a screen of 92 unrelated patients with autosomal recessive retinitis pigmentosa for defects in the human homologue of this gene. We identified seven different mutations that cosegregate with the disease. They were found among four patients with each patient heterozygously carrying two mutations. All of these mutations are predicted to affect the putative catalytic domain, probably leading to a decrease in phosphodiesterase activity and an increase in cGMP levels within rod photoreceptors. Mutations in the gene encoding the beta subunit of rod phosphodiesterase are the most common identified cause of autosomal recessive retinitis pigmentosa, accounting for approximately 4% of cases in North America.

Mutations in the key rod phototransduction enzyme phosphodiesterase 6 (PDE6) are known to cause recessive retinitis pigmentosa in humans. Mouse models of mutant PDE6 represent a common approach to understanding the mechanisms of visual disorders related to PDE6 defects. Mutation N605S in the PDE6B subunit is linked to atypical retinal degeneration 3 (atrd3) in mice. We examined PDE6 in atrd3 mice and an atrd3 mutant counterpart of human cone PDE6C expressed in rods of transgenic Xenopus laevis. These animal models revealed remarkably different phenotypes. In contrast to dramatic downregulation of the mutant rod PDE6 protein and activity levels in mice, expression and localization of the cone PDE6C in X. laevis were essentially unaffected by this mutation. Examination of the PDE6B mRNA in atrd3 retina showed that the mutation-carrying exon 14 was spliced-out in the majority of the transcript. Thus, retinal degeneration in atrd3 mice is caused by low levels of PDE6 protein due to defective processing of PDE6B pre-mRNA rather than by deleterious effects of the N605S mutation on PDE6 folding, stability or function.

Retinitis pigmentosa is an incurable retinal disease that leads to blindness. One puzzling aspect concerns the progression of the disease. Although most mutations that cause retinitis pigmentosa are in rod photoreceptor–specific genes, cone photoreceptors also die as a result of such mutations. To understand the mechanism of non-autonomous cone death, we analyzed four mouse models harboring mutations in rod-specific genes. We found changes in the insulin/mammalian target of rapamycin pathway that coincided with the activation of autophagy during the period of cone death. We increased or decreased the insulin level and measured the survival of cones in one of the models. Mice that were treated systemically with insulin had prolonged cone survival, whereas depletion of endogenous insulin had the opposite effect. These data suggest that the non-autonomous cone death in retinitis pigmentosa could, at least in part, be a result of the starvation of cones. Retinitis pigmentosa is a type of inherited retinal degeneration. It is currently untreatable and usually leads to blindness. With over 40 reintitis pigmentosa genes identified, it is the most common type of retinal degeneration caused by a single disease allele (RetNet, http://www.sph.uth.tmc.edu/Retnet/). The phenotype is characterized by an initial loss of night vision as a result of the malfunction and death of rod photoreceptors. This phase is followed by a progressive loss of cones. Because cones are responsible for color and high-acuity vision, it is their loss that leads to a reduction in the quality of life. In many cases, the disease-causing allele is expressed exclusively in rods; nonetheless, cones die as well. Indeed, to date there is no known form of retinal degeneration in humans or mice where rods die and cones survive. In contrast, mutations in cone-specific genes result only in cone death. Several theories have been proposed to explain this finding. For example, cone death could be a result of the release of a toxin produced by dying rods or the loss of a trophic factor that is produced by healthy rods 1‐6 . Alternatively, cone death could be caused by microglia that are mobilized initially during rod death 7 or by oxidative stress 8,9 .O xidative stress might also directly harm cones. The constant flow of oxygen through the retinal pigmented epithelium (RPE) to photoreceptors and the loss of rods, which are 95% of the photoreceptors in human and mouse, may result in an overload of oxygen to the remaining cones 10 . Evidence for all of these mechanisms exists in mice, yet none are able to fully explain why cones may survive for many years in the absence of rods in humans. Nonetheless, rodents are a very good model for this type of retinal degeneration. Although the rodent retina lacks a macula, which is the cone-rich and rod-free area that is present in humans, the macula is not involved in the initial phase of the disease. In humans, retinitis pigmentosa starts outside of the macula, where the distribution of rods and cones is similar to that in mice. To determine the common underlying mechanism for cone death in retinitis pigmentosa, we compared four mouse models harboring mutations in rod-specific genes (Pde6b ‐/‐ (ref. 11), Pde6g ‐/‐ (ref. 12), Rho ‐/‐ (ref. 13) and P23H 14 , which carries a Rho transgene that has an amino acid subsitution of histidine for proline at amino acid 23, as occurs in some human cases of retinitis pigmentosa, see Methods). Affymetrix arrays were used to identify common changes in gene expression that accompany cone death. Changes in a substantial number of genes involved in cellular metabolism coincided with the onset of cone death. These changes were suggestive of cones suffering from a shortage of nutrients. We then found that cones showed signs of autophagy, a cellular self-digestion process, which is consistent with prolonged starvation. We also found that several aspects of the insulin/ mammalian target of rapamycin (mTOR) pathway, an important pathway that regulates cellular metabolism, were affected during the period of cone degeneration. As a result of this finding, we increased and decreased the insulin level and measured the survival of cones in one of the models. Mice treated systemically with insulin had prolonged cone survival, whereas depletion of endogenous insulin had the opposite effect. Therefore, cone starvation is a likely contributor to the slow demise of cones in humans with retinitis pigmentosa. Treatments aimed at improving nutrition of cones are thus a plausible therapeutic avenue. RESULTS Photoreceptor death kinetics and microarray analysis To establish a framework for comparing gene expression in four different models of retinitis pigmentosa, we established the equivalent stages of disease pathology through examination of the kinetics of rod (Fig. 1 and Supplementary Figs. 1 and 2 online) and cone (Fig. 2 and

MICE homozygous for the rd mutation display hereditary retinal degeneration and the classic rd lines serve as a model for human retinitis pigmentosa1,2. In affected animals the retinal rod photo-receptor cells begin degenerating at about postnatal day 8, and by four weeks no photoreceptors are left3. Degeneration is preceded by accumulation of cyclic GMP in the retina4 and is correlated with deficient activity of the rod photoreceptor cGMP-phospho-diesterase5. We have recently isolated a candidate complementary DNA for the rd gene6 from a mouse retinal library and completed the characterization of cDNAs encoding all subunits of bovine photoreceptor phosphodiesterase7. The candidate cDNA shows strong homo logy with a cDNA encoding the bovine phospho-diesterase β subunit. Here we present evidence that the candidate cDNA is the murine homologue of bovine phosphodiesterase β cDNA. We conclude that the mouse rd locus encodes the rod photoreceptor cGMP-phosphodiesterase β subunit.

Purpose: “Sighted C3H” mice with a normal Pde6b gene, were compared to wild-type C3H/HeH mice with the strain-specific Pde6b(rd1) mutation causing retinal degeneration, BALB/c and C57BL/6 mice. Methods: All mice were screened for morphological and visual alterations by funduscopy, slit lamp biomicroscopy, laser interference biometry, ERG and optokinetic drum. Results: In “sighted C3H” mice the mutated Pde6b(rd1) allele of C3H/HeH mice was replaced by the normal Pde6b allele from BALB/c mice. The presence of the normal Pde6b gene was verified by PCR analysis. Slit lamp biomicroscopy revealed no abnormalities in the anterior segments of the eye in all mice. In the fundus of C3H/HeH (Pde6b-/-) mice the typical pigment patches and vessel attenuation caused by the retinal degeneration were observed, while the “sighted C3H” (Pde6b+/+) mice showed a normal fundus appearance, comparable to C57BL/6 mice. The albino BALB/c mice had a red-shining fundus, due to the lack of pigmentation. Scotopic ERG was performed with our screening setup for mice, where BALB/c and C57BL/6 mice showed good responses with well developed a- and b-waves, while C3H/HeH (Pde6b-/-) mice showed no response. Unexpectedly, all “sighted C3H” (Pde6b+/+) mice did not show any b-wave response in the scotopic ERG, but scored at least once within 6 trials in the optokinetic drum. As expected C3H/HeH (Pde6b-/-) mice did not respond to the moving stripes of the optokinetc drum, as did the BALB/c mice. A good response to the rotating drum was obtained from C57BL/6 mice. Conclusions: "Sighted C3H" mice have a normal Pde6b gene and responded to the rotating stripes in the optokinetc drum, but do not have normal vision due to the lack of the b-wave in the ERG. This work is supported by the NGFN.

Mutations in the beta-subunit of cGMP-phosphodiesterase (PDE6beta) can lead to either progressive retinal disease, such as human retinitis pigmentosa (RP), or stationary disease, such as congenital stationary night blindness (CSNB). Individuals with CSNB in the Rambusch pedigree were found to carry the H258N allele of PDE6B (MIM# 180072); a similar mutation was not found in RP patients. This report describes an individual carrying the H258N allele, who presented with generalized retinal dysfunction affecting the rod system and a locus of dysfunction at the rod-bipolar interface. Also described are preclinical studies in which transgenic mice with the H258N allele were generated to study the pathophysiological mechanisms of CSNB. While Pde6b(rd1)/Pde6b(rd1) mice have severe photoreceptor degeneration, as in human RP, the H258N transgene rescued these cells. The cGMP-PDE6 activity of dark-adapted H258N mice showed an approximate three-fold increase in the rate of retinal cGMP hydrolysis: from 130.1 nmol x min(-1) x nmol(-1) rhodopsin in wild-type controls to 319.2 nmol x min(-1) x nmol(-1) rhodopsin in mutants, consistent with the hypothesis that inhibition of the PDE6beta activity by the regulatory PDE6gamma subunit is blocked by this mutation. In the albino (B6CBA x FVB) F2 hybrid background, electroretinograms (ERG) from H258N mice were similar to those obtained from affected Rambusch family members, as well as humans with the most common form of CSNB (X-linked), demonstrating a selective loss of the b-wave with relatively normal a-waves. When the H258N allele was introduced into the DBA background, there was no evidence of selective reduction in b-wave amplitudes; rather a- and b-wave amplitudes were both reduced. Thus, factors other than the PDE6B mutation itself could contribute to the variance of an electrophysiological response. Therefore, caution is advisable when interpreting physiological phenotypes associated with the same allele on different genetic backgrounds. Nevertheless, such animals should be of considerable value in further studies of the molecular pathology of CSNB.

Retinal degeneration 10 (rd10) mice are a model of autosomal recessive retinitis pigmentosa (RP), identified by Chang et al. in 2002 (Vision Res. 42:517-525). These mice carry a spontaneous mutation of the rod-phosphodiesterase (PDE) gene, leading to a rod degeneration that starts around P18. Later, cones are also lost. Because photoreceptor degeneration does not overlap with retinal development, and light responses can be recorded for about a month after birth, rd10 mice mimic typical human RP more closely than the well-known rd1 mutants. The aim of this study is to provide a comprehensive analysis of the morphology and function of the rd10 mouse retina during the period of maximum photoreceptor degeneration, thus contributing useful data for exploiting this novel model to study RP. We analyzed the morphology and survival of retinal cells in rd10 mice of various ages with quantitative immunocytochemistry and confocal microscopy; we also studied retinal function with the electroretinogram (ERG), recorded between P18 and P30. We found that photoreceptor death (peaking around P25) is accompanied and followed by dendritic retraction in bipolar and horizontal cells, which eventually undergo secondary degeneration. ERG reveals alterations in the physiology of the inner retina as early as P18 (before any obvious morphological change of inner neurons) and yet consistently with a reduced band amplification by bipolar cells. Thus, changes in the rd10 retina are very similar to what was previously found in rd1 mutants. However, an overall slower decay of retinal structure and function predicts that rd10 mice might become excellent models for rescue approaches.