Rpgrip1 is required for rod outer segment development and ciliary protein trafficking in zebrafish

The authors would like to thank the Royal Society of London, the National Eye Research Centre, the Visual Research Trust, Fight for Sight, the W.H. Ross Foundation, the Rosetrees Trust, and the Glasgow Children’s Hospital Charity for supporting this work. This work was also supported by the Deanship of Scientific Research at King Saud University for funding this research (Research Project) grant number ‘RGP – VPP – 219’.Mutations in the RPGR-interacting protein 1 (RPGRIP1) gene cause recessive Leber congenital amaurosis (LCA), juvenile retinitis pigmentosa (RP) and cone-rod dystrophy. RPGRIP1 interacts with other retinal disease-causing proteins and has been proposed to have a role in ciliary protein transport; however, its function remains elusive. Here, we describe a new zebrafish model carrying a nonsense mutation in the rpgrip1 gene. Rpgrip1homozygous mutants do not form rod outer segments and display mislocalization of rhodopsin, suggesting a role for RPGRIP1 in rhodopsin-bearing vesicle trafficking. Furthermore, Rab8, the key regulator of rhodopsin ciliary trafficking, was mislocalized in photoreceptor cells of rpgrip1 mutants. The degeneration of rod cells is early onset, followed by the death of cone cells. These phenotypes are similar to that observed in LCA and juvenile RP patients. Our data indicate RPGRIP1 is necessary for rod outer segment development through regulating ciliary protein trafficking. The rpgrip1 mutant zebrafish may provide a platform for developing therapeutic treatments for RP patients.Publisher PDFPeer reviewe

Rescue of Photoreceptor Degeneration by Curcumin in Transgenic Rats with P23H Rhodopsin Mutation

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects

\u3cem\u3eRPGRIP1\u3c/em\u3e and Cone-Rod Dystrophy in Dogs

Cone–rod dystrophies (crd) represent a group of progressive inherited blinding diseases characterized by primary dysfunction and loss of cone photoreceptors accompanying or preceding rod death. Recessive crd type 1 was described in dogs associated with an RPGRIP1 exon 2 mutation, but with lack of complete concordance between genotype and phenotype. This review highlights role of the RPGRIP1, a component of complex protein networks, and its function in the primary cilium, and discusses the potential mechanisms of genotype–phenotype discordance observed in dogs with the RPGRIP1 mutation

Microglia activation in a model of retinal degeneration and TUDCA neuroprotective effects

Background: Retinitis pigmentosa is a heterogeneous group of inherited neurodegenerative retinal disorders characterized by a progressive peripheral vision loss and night vision difficulties, subsequently leading to central vision impairment. Chronic microglia activation is associated with various neurodegenerative diseases including retinitis pigmentosa. The objective of this study was to quantify microglia activation in the retina of P23H rats, an animal model of retinitis pigmentosa, and to evaluate the therapeutic effects of TUDCA (tauroursodeoxycholic acid), which has been described as a neuroprotective compound. Methods: For this study, homozygous P23H line 3 and Sprague-Dawley (SD) rats were injected weekly with TUDCA (500 mg/kg, ip) or vehicle (saline) from 20 days to 4 months old. Vertical retinal sections and whole-mount retinas were immunostained for specific markers of microglial cells (anti-CD11b, anti-Iba1 and anti-MHC-II). Microglial cell morphology was analyzed and the number of retinal microglial was quantified. Results: Microglial cells in the SD rat retinas were arranged in regular mosaics homogenously distributed within the plexiform and ganglion cell layers. In the P23H rat retina, microglial cells increased in number in all layers compared with control SD rat retinas, preserving the regular mosaic distribution. In addition, a large number of amoeboid CD11b-positive cells were observed in the P23H rat retina, even in the subretinal space. Retinas of TUDCA-treated P23H animals exhibited lower microglial cell number in all layers and absence of microglial cells in the subretinal space. Conclusions: These results report novel TUDCA anti-inflammatory actions, with potential therapeutic implications for neurodegenerative diseases, including retinitis pigmentosa.This research was supported by grants from the Spanish Ministry of Economy and Competitiveness-FEDER (BFU2012-36845), Instituto de Salud Carlos III (RETICS RD12/0034/0010), Organización Nacional de Ciegos Españoles (ONCE), FUNDALUCE, Asociación Retina Asturias and Fundación Jesús de Gangoiti

Low penetrance of retinoblastoma for p.V654L mutation of the RB1 gene

<p>Abstract</p> <p>Background</p> <p>Retinoblastoma is caused by compound heterozygosity or homozygosity of retinoblastoma gene (<it>RB1</it>) mutations. In germline retinoblastoma, mutations in the <it>RB1 </it>gene predispose individuals to increased cancer risks during development. These mutations segregate as autosomal dominant traits with high penetrance (90%).</p> <p>Methods</p> <p>We screened 30 family members from one family using high resolution melting assay and DNA direct sequencing for mutations in the <it>RB1 </it>gene. We evaluate the phenotype and penetrance of germline mutations of the <it>RB1 </it>gene in a large Taiwanese family.</p> <p>Results</p> <p>The molecular analysis and clinical details of this family showed phenotypic variability associated with the p.V654L mutation in exon 19 of the <it>RB1 </it>gene in 11 family members. The phenotype varied from asymptomatic to presence of a unilateral tumor. Only four individuals (2 males and 2 females) developed unilateral retinoblastoma, which resulted in calculated low penetrance of 36% (4/11). The four individuals with retinoblastoma were diagnosed before the age of three years. None of their relatives exhibited variable severity or bilateral retinoblastoma.</p> <p>Conclusions</p> <p>The diseased-eye ratio for this family was 0.36, which is lower than current estimates. This suggests that the <it>RB1 </it>p.V654L mutation is a typical mutation associated with low penetrance.</p

DNA testing and domestic dogs

There are currently about 80 different DNA tests available for mutations that are associated with inherited disease in the domestic dog, and as the tools available with which to dissect the canine genome become increasingly sophisticated, this number can be expected to rise dramatically over the next few years. With unrelenting media pressure focused firmly on the health of the purebred domestic dog, veterinarians and dog breeders are turning increasingly to DNA tests to ensure the health of their dogs. It is ultimately the responsibility of the scientists who identify disease-associated genetic variants to make sensible choices about which discoveries are appropriate to develop into commercially available DNA tests for the lay dog breeder, who needs to balance the need to improve the genetic health of their breed with the need to maintain genetic diversity. This review discusses some of the factors that should be considered along the route from mutation discovery to DNA test and some representative examples of DNA tests currently available

Separation of Anti-Proliferation and Anti-Apoptotic Functions of Retinoblastoma Protein through Targeted Mutations of Its A/B Domain

BACKGROUND: The human retinoblastoma susceptibility gene encodes a nuclear phosphoprotein RB, which is a negative regulator of cell proliferation. The growth suppression function of RB requires an evolutionarily conserved A/B domain that contains two distinct peptide-binding pockets. At the A/B interface is a binding site for the C-terminal trans-activation domain of E2F. Within the B-domain is a binding site for proteins containing the LxCxE peptide motif. METHODOLOGY/PRINCIPLE FINDINGS: Based on the crystal structure of the A/B domain, we have constructed an RB-K530A/N757F (KN) mutant to disrupt the E2F- and LxCxE-binding pockets. The RB-K530A (K) mutant is sufficient to inactivate the E2F-binding pocket, whereas the RB-N757F (N) mutant is sufficient to inactivate the LxCxE-binding pocket. Each single mutant inhibits cell proliferation, but the RB-KN double mutant is defective in growth suppression. Nevertheless, the RB-KN mutant is capable of reducing etoposide-induced apoptosis. CONCLUSION/SIGNIFICANCE: Previous studies have established that RB-dependent G1-arrest can confer resistance to DNA damage-induced apoptosis. Results from this study demonstrate that RB can also inhibit apoptosis independent of growth suppression

Functional Analysis of Retinitis Pigmentosa 2 (RP2) Protein Reveals Variable Pathogenic Potential of Disease-Associated Missense Variants

Genetic mutations are frequently associated with diverse phenotypic consequences, which limits the interpretation of the consequence of a variation in patients. Mutations in the retinitis pigmentosa 2 (RP2) gene are associated with X-linked RP, which is a phenotypically heterogenic form of retinal degeneration. The purpose of this study was to assess the functional consequence of disease-associated mutations in the RP2 gene using an in vivo assay. Morpholino-mediated depletion of rp2 in zebrafish resulted in perturbations in photoreceptor development and microphthalmia (small eye). Ultrastructural and immunofluorescence analyses revealed defective photoreceptor outer segment development and lack of expression of photoreceptor-specific proteins. The retinopathy phenotype could be rescued by expressing the wild-type human RP2 protein. Notably, the tested RP2 mutants exhibited variable degrees of rescue of rod versus cone photoreceptor development as well as microphthalmia. Our results suggest that RP2 plays a key role in photoreceptor development and maintenance in zebrafish and that the clinical heterogeneity associated with RP2 mutations may, in part, result from its potentially distinct functional relevance in rod versus cone photoreceptors

Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model

The authors are grateful to Manuel Simonutti, Julie Dégardin, Jennifer Da Silva, Samantha Beck and Caroline Carvalho for their valuable help in phenotyping (platform of Institut de la Vision) and to Isabelle Renault, Léa Biedermann and André Tiffoche for animal care (platform of Institut de la Vision). The authors thank Stéphane Fouquet for his support in developing a custom-made Image J macro to measure thickness of retinal layers.This work was supported by Fondation Valentin Hauy (IA, EO), Retina France (IA, EO), e-rare RHORCOD (IA), Fondation de l’Oeil—Fondation de France (IA), Foundation Voir et Entendre (CZ), Foundation Fighting Blindness (FFB) (CD-CL-0808-0466-CHNO) (IA), and the FFB center grant (CD-CL-0808-0466-CHNO), Ville de Paris and Region Ile de France, Labex Lifesenses (reference ANR-10-LABX-65) supported by French state funds managed by the ANR within the Investissements d’Avenir programme (ANR-11-IDEX-0004-0), the Regional Council of Ile de France (I09–1727/R) (EO), the National Institute of Health grants EY10609 (MIN), EY001919 (MML) and EY006842 (MML) and the Foundation Fighting Blindness (MIN and MML).Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3’ UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.Yeshttp://www.plosone.org/static/editorial#pee

Novel Expression Patterns of Metabotropic Glutamate Receptor 6 in the Zebrafish Nervous System

The metabotropic glutamate receptor 6 (mGluR6 or GRM6) belongs to the class III of the metabotropic glutamate receptor family. It is the only known mGluR that mediates direct synaptic transmission in the nervous system and is thought to mediate the ON-response in the ON-pathway of the vertebrate retina. Phylogenetic and gene structure analysis indicated that the zebrafish genome harbours two mglur6 paralogs, mglur6a and mglur6b. Besides expression in the inner nuclear layer and distinct regions in the brain, both mglur6 paralogs are expressed in ganglion cells of the retina, an expression pattern which can also be observed in the downstream effector molecules gnaoa and gnaob. This unexpected expression pattern is consistent with immunohistological labeling using a peptide antibody specific for the mGluR6b paralog. These expression patterns contradict the existing view that mGluR6 is solely located on ON-bipolar cells where it functions in signal transmission. Consistent with expression in ON-bipolar cells, we report a decreased b-wave amplitude in the electroretinogram after morpholino-based downregulation of mGluR6b, showing a function in the ON response. Our data suggest more widespread functions of mGluR6 mediated signaling in the central nervous system, possibly including sign reversing synapses in the inner retina