11-cis-retinal and Retinal-Dystrophies

Primary cilia are almost ubiquitously expressed in eukaryotic cells where they function as sensors relaying information either from the extracellular environment or between two compartments of the same cell, such as in the photoreceptor cell. In ciliopathies, a continuously growing class of genetic disorders related to ciliary defects, the modified primary cilium of the photoreceptor, also known as the connecting cilium, is frequently defective. Ciliary dysfunction involves disturbances in the trafficking and docking of specific proteins involved in its biogenesis or maintenance. The main well-conserved ciliary process, intraflagellar transport (IFT), is a complex process carried out by multimeric ciliary particles and molecular motors of major importance in the photoreceptor cell. It is defective in a growing number of ciliopathies leading to retinal degeneration. Retinitis pigmentosa related to ciliary dysfunction can be an isolated feature or a part of a syndrome such as Bardet-Biedl syndrome (BBS). Research on ciliopathies and BBS has led to the discovery of several major cellular processes carried out by the primary cilium structure and has highlighted their genetic heterogeneity.

Topics: Bardet-Biedl Syndrome; Cilia; Cognition Disorders; Humans; Models, Biological; Photoreceptor Cells, Vertebrate; Protein Transport; Retinal Dystrophies; Rhodopsin

Inherited retinal dystrophies (RDs) are heterogenous in many aspects including genes involved, age of onset, rate of progression, and treatments. While RDs are caused by a plethora of different mutations, all result in the same outcome of blindness. While treatments, both gene therapy-based and drug-based, have been developed to slow or halt disease progression and prevent further blindness, only a small handful of the forms of RDs have treatments available, which are primarily for recessively inherited forms. Using immunohistochemical methods coupled with electroretinography, optical coherence tomography, and fluorescein angiography, we show that in rhodopsin mutant mice, the involvement of both the innate and the autoimmune systems could be a strong contributing factor in disease progression and pathogenesis. Herein, we show that monocytic phagocytosis and inflammatory cytokine release along with protein citrullination, a major player in forms of autoimmunity, work to enhance the progression of RD associated with a rhodopsin mutation.

Topics: Animals; Autoimmunity; Electroretinography; Humans; Immunity, Innate; Mice; Mutation; Phenotype; Retinal Dystrophies; Rhodopsin

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. We recently identified mutations in REEP6, which encodes the receptor expression enhancing protein 6, in several families with autosomal recessive RP. REEP6 is related to the REEP and Yop1p family of ER shaping proteins and potential receptor accessory proteins, but the role of REEP6 in the retina is unknown. Here we characterize the disease mechanisms associated with loss of REEP6 function using a Reep6 knockout mouse generated by CRISPR/Cas9 gene editing. In control mice REEP6 was localized to the inner segment and outer plexiform layer of rod photoreceptors. The Reep6-/- mice exhibited progressive photoreceptor degeneration from P20 onwards. Ultrastructural analyses at P20 by transmission electron microscopy and 3View serial block face scanning EM revealed an expansion of the distal ER in the Reep6-/- rods and an increase in their number of mitochondria. Electroretinograms revealed photoreceptor dysfunction preceded degeneration, suggesting potential defects in phototransduction. There was no effect on the traffic of rhodopsin, Rom1 or peripherin/rds; however, the retinal guanylate cyclases GC1 and GC2 were severely affected in the Reep6 knockout animals, with almost undetectable expression. These changes correlated with an increase in C/EBP homologous protein (CHOP) expression and the activation of caspase 12, suggesting that ER stress contributes to cell death. Collectively, these data suggest that REEP6 plays an essential role in maintaining cGMP homeostasis though facilitating the stability and/or trafficking of guanylate cyclases and maintaining ER and mitochondrial homeostasis.

Topics: Animals; Base Sequence; Clustered Regularly Interspaced Short Palindromic Repeats; Endoplasmic Reticulum; Eye Proteins; Gene Editing; Guanylate Cyclase; Light Signal Transduction; Membrane Proteins; Membrane Transport Proteins; Mice; Mice, Knockout; Photoreceptor Cells, Vertebrate; Retinal Dystrophies; Retinal Rod Photoreceptor Cells; Rhodopsin

This study aimed to identify the underlying molecular genetic cause in four Spanish families clinically diagnosed of Retinitis Pigmentosa (RP), comprising one autosomal dominant RP (adRP), two autosomal recessive RP (arRP) and one with two possible modes of inheritance: arRP or X-Linked RP (XLRP). We performed whole exome sequencing (WES) using NimbleGen SeqCap EZ Exome V3 sample preparation kit and SOLID 5500xl platform. All variants passing filter criteria were validated by Sanger sequencing to confirm familial segregation and the absence in local control population. This strategy allowed the detection of: (i) one novel heterozygous splice-site deletion in RHO, c.937-2_944del, (ii) one rare homozygous mutation in C2orf71, c.1795T>C; p.Cys599Arg, not previously associated with the disease, (iii) two heterozygous null mutations in ABCA4, c.2041C>T; p.R681* and c.6088C>T; p.R2030*, and (iv) one mutation, c.2405-2406delAG; p.Glu802Glyfs*31 in the ORF15 of RPGR. The molecular findings for RHO and C2orf71 confirmed the initial diagnosis of adRP and arRP, respectively, while patients with the two ABCA4 mutations, both previously associated with Stargardt disease, presented symptoms of RP with early macular involvement. Finally, the X-Linked inheritance was confirmed for the family with the RPGR mutation. This latter finding allowed the inclusion of carrier sisters in our preimplantational genetic diagnosis program.

Topics: Adolescent; Adult; Amino Acid Sequence; Base Sequence; Child; Chromosome Segregation; DNA Mutational Analysis; Exome; Family; Female; Humans; Inheritance Patterns; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Retinal Dystrophies; Rhodopsin

Rhodopsin has been used as a prototype system to investigate G protein-coupled receptor (GPCR) internalization and endocytic sorting mechanisms. Failure of rhodopsin recycling upon light activation results in various degenerative retinal diseases. Accumulation of internalized rhodopsin in late endosomes and the impairment of its lysosomal degradation are associated with unregulated cell death that occurs in dystrophies. However, the molecular basis of rhodopsin accumulation remains elusive. We found that the novel norpA(P24) suppressor, diehard4, is responsible for the inability of endo-lysosomal rhodopsin trafficking and retinal degeneration in Drosophila models of retinal dystrophies. We found that diehard4 encodes Osiris 21. Loss of its function suppresses retinal degeneration in norpA(P24), rdgC(306), and trp(1), but not in rdgB(2), suggesting a common cause of photoreceptor death. In addition, the loss of Osiris 21 function shifts the membrane balance between late endosomes and lysosomes as evidenced by smaller late endosomes and the proliferation of lysosomal compartments, thus facilitating the degradation of endocytosed rhodopsin. Our results demonstrate the existence of negative regulation in vesicular traffic between endosomes and lysosomes. We anticipate that the identification of additional components and an in-depth description of this specific molecular machinery will aid in therapeutic interventions of various retinal dystrophies and GPCR-related human diseases.

Topics: Animals; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Endocytosis; Endosomes; Humans; Lysosomes; Membrane Proteins; Phospholipase C beta; Photoreceptor Cells, Invertebrate; Retinal Dystrophies; Rhodopsin; Vesicular Transport Proteins

Bothnia dystrophy is a variant of recessive retinitis punctata albescens (RPA) and is caused by a homozygous R234W mutation in the RLBP1 gene. We report the clinical features of a Japanese patient with the homozygous R234W mutation in the RLBP1 gene.. An affected woman with RPA has been examined clinically for 25 years. Her DNA was obtained with informed consent, and the exons and surrounding areas of RDH5, rhodopsin, and RLBP1 were amplified by PCR and directly sequenced.. Our patient was first examined in our hospital in 1986 when she was 6 years old. Ophthalmoscopy showed numerous small white dots in the posterior pole of both eyes. Although the a- and b-waves of the single flash ERGs were severely reduced after a standard 30 min of dark-adaptation, the amplitudes of both waves increased markedly after 24 hr of dark-adaptation. The visual disturbances and visual field scotomas became more evident in her twenties, and her BCVAs were 0.2 OD and 0.5 OS when she was 31 years old in 2010. Fundus examinations showed macular degeneration in both eyes. A homozygous R234W mutation was detected in RLBP1, and no mutations were detected in RDH5 and rhodopsin.. The clinical characteristics of a Japanese patient with a homozygous R234W mutation in RLBP1 are very similar to that of Swedish patients with Bothnia dystrophy. The origin of the Japanese R234W mutation is probably not the same as that of the Swedish patients, but more likely due to the high incidence of C to T transitions.

Topics: Adult; Alcohol Oxidoreductases; Asian People; Carrier Proteins; Dark Adaptation; DNA Mutational Analysis; Electroretinography; Exons; Female; Fluorescein Angiography; Humans; Japan; Mutation, Missense; Night Blindness; Polymerase Chain Reaction; Retinal Dystrophies; Rhodopsin; Tomography, Optical Coherence; Visual Acuity; Visual Fields

The P23H-1 rat strain carries a rhodopsin mutation frequently found in retinitis pigmentosa patients. We investigated the progressive degeneration of the inner retina in this strain, focussing on retinal ganglion cells (RGCs) fate. Our data show that photoreceptor death commences in the ventral retina, spreading to the whole retina as the rat ages. Quantification of the total number of RGCs identified by Fluorogold tracing and Brn3a expression, disclosed that the population of RGCs in young P23H rats is significantly smaller than in its homologous SD strain. In the mutant strain, there is also RGC loss with age: RGCs show their first symptoms of degeneration at P180, as revealed by an abnormal expression of cytoskeletal proteins which, at P365, translates into a significant loss of RGCs, that may ultimately be caused by displaced inner retinal vessels that drag and strangulate their axons. RGC axonal compression begins also in the ventral retina and spreads from there causing RGC loss through the whole retinal surface. These decaying processes are common to several models of photoreceptor loss, but show some differences between inherited and light-induced photoreceptor degeneration and should therefore be studied to a better understanding of photoreceptor degeneration and when developing therapies for these diseases.

Topics: Aging; Animals; Animals, Genetically Modified; Apoptosis; Axons; Cell Count; Cytoskeletal Proteins; Disease Models, Animal; Female; Fluorescent Antibody Technique, Indirect; Mutation; Photoreceptor Cells, Vertebrate; Rats; Retinal Dystrophies; Retinal Ganglion Cells; Rhodopsin; Stilbamidines; Transcription Factor Brn-3A

Retinal dystrophies, known in man, dog, mouse, and rat, involve progressive loss of photoreceptor cells with onset during or soon after the developmental period. Functional (electroretinogram), chemical (rhodopsin analyses) and morphological (light and electron microscopy) data obtained in the rat indicated two main processes: (a) overproduction of rhodopsin and an associated abnormal lamellar tissue component, (b) progressive loss of photoreceptor cells. The first abnormality recognized was the appearance of swirling sheets or bundles of extracellular lamellae between normally developing retinal rods and pigment epithelium; membrane thickness and spacing resembled that in normal outer segments. Rhodopsin content reached twice normal values, was present in both rods and extracellular lamellae, and was qualitatively normal, judged by absorption maximum and products of bleaching. Photoreceptors attained virtually adult form and ERG function. Then rod inner segments and nuclei began degenerating; the ERG lost sensitivity and showed selective depression of the a-wave at high luminances. Outer segments and lamellae gradually degenerated and rhodopsin content decreased. No phagocytosis was seen, though pigment cells partially dedifferentiated and many migrated through the outer segment-debris zone toward the retina. Eventually photoreceptor cells and the b-wave of the ERG entirely disappeared. Rats kept in darkness retained electrical activity, rhodopsin content, rod structure, and extracellular lamellae longer than litter mates in light.

Topics: Animals; Disease; Dogs; Electrons; Electroretinography; Humans; Light; Male; Mice; Microscopy; Microscopy, Electron; Photoreceptor Cells; Rats; Retina; Retinal Diseases; Retinal Dystrophies; Rhodopsin; Vision, Ocular