Translational readthrough as a potential therapeutic for AIPL1-associated Leber Congenital Amaurosis in a patient-derived iPSC-retinal organoid model

Leber Congenital Amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterised by severe sight impairment in infancy and rapidly progressive degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cGMP phosphodiesterase (PDE6) compared to control or CRISPR corrected organoids. Moreover, increased levels of cGMP were detected. The translational readthrough inducing drug (TRID) PTC124 was investigated as a potential therapeutic. LCA4 retinal organoids exhibited rescue of AIPL1 and PDE6; however, the level of full-length, functional AIPL1 induced through PTC124 treatment was insufficient to reduce cGMP levels and fully rescue the LCA4 phenotype. LCA4 retinal organoids are a valuable platform for the in vitro investigation of the molecular mechanisms that drive photoreceptor loss and for the evaluation of novel therapeutics.

Effect of NK-5962 on Gene Expression Profiling of Retina in a Rat Model of Retinitis Pigmentosa

Purpose: NK-5962 is a key component of photoelectric dye-coupled polyethylene film, designated Okayama University type-retinal prosthesis (OUReP™). Previously, we found that NK-5962 solution could reduce the number of apoptotic photoreceptors in the eyes of the Royal College of Surgeons (RCS) rats by intravitreal injection under a 12 h light/dark cycle. This study aimed to explore possible molecular mechanisms underlying the anti-apoptotic effect of NK-5962 in the retina of RCS rats. Methods: RCS rats received intravitreal injections of NK-5962 solution in the left eye at the age of 3 and 4 weeks, before the age of 5 weeks when the speed in the apoptotic degeneration of photoreceptors reaches its peak. The vehicle-treated right eyes served as controls. All rats were housed under a 12 h light/dark cycle, and the retinas were dissected out at the age of 5 weeks for RNA sequence (RNA-seq) analysis. For the functional annotation of differentially expressed genes (DEGs), the Metascape and DAVID databases were used. Results: In total, 55 up-regulated DEGs, and one down-regulated gene (LYVE1) were found to be common among samples treated with NK-5962. These DEGs were analyzed using Gene Ontology (GO) term enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway analyses. We focused on the up-regulated DEGs that were enriched in extracellular matrix organization, extracellular exosome, and PI3K–Akt signaling pathways. These terms and pathways may relate to mechanisms to protect photoreceptor cells. Moreover, our analyses suggest that SERPINF1, which encodes pigment epithelium-derived factor (PEDF), is one of the key regulatory genes involved in the anti-apoptotic effect of NK-5962 in RCS rat retinas. Conclusions: Our findings suggest that photoelectric dye NK-5962 may delay apoptotic death of photoreceptor cells in RCS rats by up-regulating genes related to extracellular matrix organization, extracellular exosome, and PI3K–Akt signaling pathways. Overall, our RNA-seq and bioinformatics analyses provide insights in the transcriptome responses in the dystrophic RCS rat retinas that were induced by NK-5962 intravitreal injection and offer potential target genes for developing new therapeutic strategies for patients with retinitis pigmentosa.

Importance of Autoimmune Responses in Progression of Retinal Degeneration Initiated by Gene Mutations

Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous rare disorders associated with retinal dysfunction and death of retinal photoreceptor cells, leading to blindness. Among the most frequent and severe forms of those retinopathies is retinitis pigmentosa (RP) that affects 1:4,000 individuals worldwide. The genes that have been implicated in RP are associated with the proteins present in photoreceptor cells or retinal pigment epithelium (RPE). Asymmetric presentation or sudden progression in retinal disease suggests that a gene mutation alone might not be responsible for retinal degeneration. Immune responses could directly target the retina or be site effect of immunity as a bystander deterioration. Autoantibodies against retinal autoantigens have been found in RP, which led to a hypothesis that autoimmunity could be responsible for the progression of photoreceptor cell death initiated by a genetic mutation. The other contributory factor to retinal degeneration is inflammation that activates the innate immune mechanisms, such as complement. If autoimmune responses contribute to the progression of retinopathy, this could have an implication on treatment, such as gene replacement therapy. In this review, we provide a perspective on the current role of autoimmunity/immunity in RP pathophysiology.

A computational pipeline for functional gene discovery

Many computational pipelines exist for the detection of differentially expressed genes. However, computational pipelines for functional gene detection rarely exist. We developed a new computational pipeline for functional gene identification from transcriptome profiling data. Key features of the pipeline include batch effect correction, clustering optimization by gap statistics, gene ontology analysis of clustered genes, and literature analysis for functional gene discovery. By leveraging this pipeline on RNA-seq datasets from two mouse retinal development studies, we identified 7 candidate genes involved in the formation of the photoreceptor outer segment. The expression of top three candidate genes (Pde8b, Laptm4b, and Nr1h4) in the outer segment of the developing mouse retina were experimentally validated by immunohistochemical analysis. This computational pipeline can accurately predict novel functional gene for a specific biological process, e.g., development of the outer segment and synapses of the photoreceptor cells in the mouse retina. This pipeline can also be useful to discover functional genes for other biological processes and in other organs and tissues.

The rhodopsin-retinochrome system for retinal re-isomerization predates the origin of cephalopod eyes

Background The process of photoreception in most animals depends on the light induced isomerization of the chromophore retinal, bound to rhodopsin. To re-use retinal, the all-trans-retinal form needs to be re-isomerized to 11-cis-retinal, which can be achieved in different ways. In vertebrates, this mostly includes a stepwise enzymatic process called the visual cycle. The best studied re-isomerization system in protostomes is the rhodopsin-retinochrome system of cephalopods, which consists of rhodopsin, the photoisomerase retinochrome and the protein RALBP functioning as shuttle for retinal. In this study we investigate the expression of the rhodopsin-retinochrome system and functional components of the vertebrate visual cycle in a polyplacophoran mollusk, Leptochiton asellus, and examine the phylogenetic distribution of the individual components in other protostome animals. Results Tree-based orthology assignments revealed that orthologs of the cephalopod retinochrome and RALBP are present in mollusks outside of cephalopods. By mining our dataset for vertebrate visual cycle components, we also found orthologs of the retinoid binding protein RLBP1, in polyplacophoran mollusks, cephalopods and a phoronid. In situ hybridization and antibody staining revealed that L. asellus retinochrome is co-expressed in the larval chiton photoreceptor cells (PRCs) with the visual rhodopsin, RALBP and RLBP1. In addition, multiple retinal dehydrogenases are expressed in the PRCs, which might also contribute to the rhodopsin-retinochrome system. Conclusions We conclude that the rhodopsin-retinochrome system is a common feature of mollusk PRCs and predates the origin of cephalopod eyes. Our results show that this system has to be extended by adding further components, which surprisingly, are shared with vertebrates.

Protein Kinase A in Human Retina: Differential Localization of Cβ, Cα, RIIα, and RIIβ in Photoreceptors Highlights Non-redundancy of Protein Kinase A Subunits

Protein kinase A (PKA) signaling is essential for numerous processes but the subcellular localization of specific PKA regulatory (R) and catalytic (C) subunits has yet to be explored comprehensively. Additionally, the localization of the Cβ subunit has never been spatially mapped in any tissue even though ∼50% of PKA signaling in neuronal tissues is thought to be mediated by Cβ. Here we used human retina with its highly specialized neurons as a window into PKA signaling in the brain and characterized localization of PKA Cα, Cβ, RIIα, and RIIβ subunits. We found that each subunit presented a distinct localization pattern. Cα and Cβ were localized in all cell layers (photoreceptors, interneurons, retinal ganglion cells), while RIIα and RIIβ were selectively enriched in photoreceptor cells where both showed distinct patterns of co-localization with Cα but not Cβ. Only Cα was observed in photoreceptor outer segments and at the base of the connecting cilium. Cβ in turn, was highly enriched in mitochondria and was especially prominent in the ellipsoid of cone cells. Further investigation of Cβ using RNA BaseScope technology showed that two Cβ splice variants (Cβ4 and Cβ4ab) likely code for the mitochondrial Cβ proteins. Overall, our data indicates that PKA Cα, Cβ, RIIα, and RIIβ subunits are differentially localized and are likely functionally non-redundant in the human retina. Furthermore, Cβ is potentially important for mitochondrial-associated neurodegenerative diseases previously linked to PKA dysfunction.

Cone photoreceptor dysfunction in retinitis pigmentosa revealed by optoretinography

Retinitis pigmentosa (RP) is the most common group of inherited retinal degenerative diseases, whose most debilitating phase is cone photoreceptor death. Perimetric and electroretinographic methods are the gold standards for diagnosing and monitoring RP and assessing cone function. However, these methods lack the spatial resolution and sensitivity to assess disease progression at the level of individual photoreceptor cells, where the disease originates and whose degradation causes vision loss. High-resolution retinal imaging methods permit visualization of human cone cells in vivo but have only recently achieved sufficient sensitivity to observe their function as manifested in the cone optoretinogram. By imaging with phase-sensitive adaptive optics optical coherence tomography, we identify a biomarker in the cone optoretinogram that characterizes individual cone dysfunction by stimulating cone cells with flashes of light and measuring nanometer-scale changes in their outer segments. We find that cone optoretinographic responses decrease with increasing RP severity and that even in areas where cone density appears normal, cones can respond differently than those in controls. Unexpectedly, in the most severely diseased patches examined, we find isolated cones that respond normally. Short-wavelength–sensitive cones are found to be more vulnerable to RP than medium- and long-wavelength–sensitive cones. We find that decreases in cone response and cone outer-segment length arise earlier in RP than changes in cone density but that decreases in response and length are not necessarily correlated within single cones.

Neuroinflammation as a Therapeutic Target in Retinitis Pigmentosa and Quercetin as Its Potential Modulator

The retina is a multilayer neuronal tissue located in the back of the eye that transduces the environmental light into a neural impulse. Many eye diseases caused by endogenous or exogenous harm lead to retina degeneration with neuroinflammation being a major hallmark of these pathologies. One of the most prevalent retinopathies is retinitis pigmentosa (RP), a clinically and genetically heterogeneous hereditary disorder that causes a decline in vision and eventually blindness. Most RP cases are related to mutations in the rod visual receptor, rhodopsin. The mutant protein triggers inflammatory reactions resulting in the activation of microglia to clear degenerating photoreceptor cells. However, sustained insult caused by the abnormal genetic background exacerbates the inflammatory response and increases oxidative stress in the retina, leading to a decline in rod photoreceptors followed by cone photoreceptors. Thus, inhibition of inflammation in RP has received attention and has been explored as a potential therapeutic strategy. However, pharmacological modulation of the retinal inflammatory response in combination with rhodopsin small molecule chaperones would likely be a more advantageous therapeutic approach to combat RP. Flavonoids, which exhibit antioxidant and anti-inflammatory properties, and modulate the stability and folding of rod opsin, could be a valid option in developing treatment strategies against RP.

Cryptochrome magnetoreception: four tryptophans could be better than three

The biophysical mechanism of the magnetic compass sensor in migratory songbirds is thought to involve photo-induced radical pairs formed in cryptochrome (Cry) flavoproteins located in photoreceptor cells in the eyes. In Cry4a—the most likely of the six known avian Crys to have a magnetic sensing function—four radical pair states are formed sequentially by the stepwise transfer of an electron along a chain of four tryptophan residues to the photo-excited flavin. In purified Cry4a from the migratory European robin, the third of these flavin–tryptophan radical pairs is more magnetically sensitive than the fourth, consistent with the smaller separation of the radicals in the former. Here, we explore the idea that these two radical pair states of Cry4a could exist in rapid dynamic equilibrium such that the key magnetic and kinetic properties are weighted averages. Spin dynamics simulations suggest that the third radical pair is largely responsible for magnetic sensing while the fourth may be better placed to initiate magnetic signalling particularly if the terminal tryptophan radical can be reduced by a nearby tyrosine. Such an arrangement could have allowed independent optimization of the essential sensing and signalling functions of the protein. It might also rationalize why avian Cry4a has four tryptophans while Crys from plants have only three.